translational control of viral gene expression in …translational control of viral gene expression...

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS,1092-2172/00/$04.0010

June 2000, p. 239–280 Vol. 64, No. 2

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Translational Control of Viral Gene Expression in EukaryotesMICHAEL GALE, JR.,1* SENG-LAI TAN,2 AND MICHAEL G. KATZE2

University of Texas Southwestern Medical Center, Dallas, Texas,1 andUniversity of Washington, Seattle, Washington2

INTRODUCTION .......................................................................................................................................................240Overview of Eukaryotic mRNA Translation and Sites of Viral Regulation ...................................................240

Viral translational programming .....................................................................................................................240Translation initiation .........................................................................................................................................241Cap-binding reaction..........................................................................................................................................241Ribosome scanning and AUG site selection....................................................................................................242Elongation............................................................................................................................................................243Termination .........................................................................................................................................................243Improving translation efficiency: the closed-loop model of mRNA translation .........................................243

TRANSLATIONAL CONTROL OF VIRAL GENE EXPRESSION ....................................................................243Advantages and liabilities of cap-dependent host translation .....................................................................243

Host Shutoff and Selective Translation of Viral mRNA....................................................................................244Mechanisms and Control of Viral mRNA Translation .....................................................................................245

Internal ribosome entry .....................................................................................................................................245Ribosome shunt...................................................................................................................................................248Leaky scanning....................................................................................................................................................249Frameshifting ......................................................................................................................................................249Control of termination and reinitiation ..........................................................................................................251Functional recoding ............................................................................................................................................252

Coupling the Virus Life Cycle to Translational Control...................................................................................252The herpesviruses ...............................................................................................................................................253HSV and the shutoff of host cell protein synthesis........................................................................................253Selective repression of mRNA translation initiation during HSV infection ..............................................254Selective translation of HSV mRNA ................................................................................................................254Inhibition of eIF2a phosphorylation during HSV infection.........................................................................255Implications of viral modulation of translation in HSV pathogenesis and disease treatment ...............256

Recruitment of Host Factors for the Efficient Translation of Viral mRNA...................................................256IRES binding proteins and proteins that bind the viral 3* UTR ................................................................256Influenza virus.....................................................................................................................................................258Selective translation of influenza virus mRNAs.............................................................................................258Contribution of influenza virus mRNA structure upon selective translation ............................................258Influenza virus recruitment of Grsf-1..............................................................................................................259Temporal regulation of influenza virus mRNA translation..........................................................................260Maintenance of translation in influenza virus-infected cells .......................................................................260Recruitment of P58IPK and inhibition of eIF2a phosphorylation during influenza virus infection.......260Role of the influenza virus NS1 protein in viral mRNA translation...........................................................261

VIRAL MODIFICATION OF CELLULAR FACTORS .........................................................................................262Inactivation of eIF4E and modulation of the eIF4E-binding proteins........................................................262Cleavage of eIF4G...............................................................................................................................................263Cleavage of PABP: disruption of the closed-loop translation complex.......................................................263Modification of EF-1 ..........................................................................................................................................264Disruption of eIF2a phosphorylation ..............................................................................................................264

(i) PKR structure and function ....................................................................................................................264(ii) Mechanisms of PKR inhibition by eukaryotic viruses........................................................................265

Disruption of the IFN-induced cellular antiviral response through inhibition of PKR...........................266(i) Viral inhibition of PKR: HCV .................................................................................................................266

VIRAL PERSISTENCE AND TRANSLATIONAL CONTROL............................................................................266Translational programming and maintenance of viral persistence.............................................................267Translational control, persistent infection, and regulation of host apoptosis ...........................................267Cell growth control, eIF2a phosphorylation, and oncogenic transformation ............................................268

* Corresponding author. Mailing address: Department of Microbi-ology, University of Texas Southwestern Medical Center, 5323 HarryHines Blvd., Dallas, TX 75390-9048. Phone: (214) 648-5940. Fax: (214)648-5905. E-mail: [email protected].

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mRNA TRANSLATION AS A TARGET FOR ANTIVIRAL THERAPY .............................................................268CONCLUSIONS AND PERSPECTIVES.................................................................................................................269ACKNOWLEDGMENTS ...........................................................................................................................................270REFERENCES ............................................................................................................................................................270

INTRODUCTION

Perhaps nowhere in nature is a parasitic relationship as welldefined as that which occurs between a virus and its host cell.Viruses rely on the host cell for propagation, utilizing cellularmachinery for the replication and assembly of viral compo-nents and the release of progeny virions. Whether possessing aDNA or RNA genome, the eukaryotic virus exhibits a generallife cycle that is initiated through interaction with its cognatereceptor(s) on the surface of the host cell (117) (Fig. 1). Aftervirion adsorption and internalization, uncoating exposes theviral genome and associated proteins to the host milieu, where-upon genome replication and transcription take place. Thetranslation of viral RNA is followed by the assembly of struc-tural proteins, packaging of the viral genome, and eventualrelease of progeny virions. While some viruses encode or carrythe enzymatic machinery required for autonomous genomereplication and/or transcription, others recruit host poly-merases to carryout this task (117). In contrast, viruses donot encode or carry the machinery for mRNA translation.Thus, the ensuing stage of viral protein synthesis is com-pletely dependent on the translational machinery of the hostcell (Fig. 1). Not surprisingly, viruses have devoted muchattention to this dependency and have evolved strategiesthat reduce the impact of translational dependence on viralreplication. As discussed in this review, these strategies arethemselves limited by the nature of the viral RNA, the cellulartranslation machinery, and the translation regulatory pathwaysof the host cell.

This treatise presents an overview of translation strategiesused by viruses that infect the cells of higher eukaryotes.Where appropriate, we have focused on specific virus systemsto present examples of the diverse mechanisms by which vi-ruses overcome the problems of translational dependence. Forcomplementary material on mRNA translation, virus-host in-teractions, host antiviral pathways, and the virus-host interac-tions of lower eukaryotes and bacteria, we direct the reader toseveral fine texts and reviews (1, 10, 117, 137, 179, 199, 303,324, 353, 415, 416, 428, 455, 480, 484). We begin this reviewwith a brief overview of the current models for eukaryoticmRNA translation, including points of translational control,and effects on host translation due to virus infection. This isfollowed by examples of translation strategies that are depen-dent on the structure of the viral mRNA and those that aredirected at recruitment and modification of the translationmachinery and other host factors. Given the recent emphasis inthe translational control field on identifying and characterizingcellular signaling pathways that govern mRNA translation (43,120, 433), we have included discussion of how viruses mightexploit these pathways to facilitate completion of their trans-lational programs. Attention is directed to the ways in whichdisruption of host translational control pathways may contrib-ute to viral pathogenesis and disease progression. Finally, weconclude with a section describing the prospects of targetingviral mRNA translation for antiviral therapy, as well as per-spectives for future research in the increasingly overlappingdisciplines of virology, viral pathogenesis, and translation con-trol.

Overview of Eukaryotic mRNA Translation and Sitesof Viral Regulation

Translation in eukaryotes is a complex multistep, multipro-tein process (198, 335). As with most complex biochemicalreactions, it is subject to strict regulatory controls, and is ex-tremely sensitive to both the intracellular and extracellularenvironments (43, 163, 232, 281, 324, 433). In general, thetranslation of a given mRNA can be modulated in response tonutrient availability, mitogenic stimulation and cell cycle reg-ulation, stress, and, as described herein, viral infection (re-viewed in detail in reference 199). It is also increasingly clearfrom research spanning the past several years that regulationof mRNA translation is critical for maintaining control of cellgrowth (96, 287, 429). As presented in the “Viral persistenceand translational control” section (below), disruption of themajor translation checkpoints and signaling cascades renderscells unable to respond to translation-modulatory signals andmay constitute a mechanism of oncogenic transformation (67).The following sections provide a general overview of viraltranslational programming and eukaryotic mRNA translation.Major sites for virus regulation of translation are noted, andthey are discussed in detail in this review.

Viral translational programming. Viruses face enormouspressures to maintain a “functional” genome size, which great-

FIG. 1. General model of eukaryotic viral replication. Viral particles areshown in black. Viruses recognize their target cell through interaction withspecific receptors and/or other components on the cell membrane. Interactionwith the host cell induces cell membrane penetration and virion internalization.Virion uncoating releases the viral genome, whereupon it is available for tran-scription and translation. Poxviruses and the RNA viruses (with the exception ofretroviruses) replicate in the cytoplasm. Transcription of all other DNA virusestakes place in the nucleus. Transcription and genome replication are followed bythe cytoplasmic stages of mRNA translation and virion assembly. Release ofmature virions may include membrane lysis and death of the host cell. Adaptedwith modification from reference 322.

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ly influences the rate and efficiency of viral replication. Thus,host translational dependence may in part reflect the limita-tions placed on viral replication due to the enormous genomecapacity that would be needed to encode the components forautonomous viral protein synthesis (335). This idea is sup-ported by the highly specialized nature of the protein syntheticmachinery, which encompasses well over 30 different geneproducts and yet remains highly conserved between the pro-karyotic and eukaryotic kingdoms (335, 470). Eukaryotic vi-ruses have evolved effective means of exploiting their innatetranslational dependence through mechanisms of translationalprogramming. This is the process in which eukaryotic viruses(i) redirect the host translation machinery to favor viral proteinsynthesis and (ii) control the expression of their own geneproducts. The latter is especially important for the RNA vi-ruses, which have limited transcriptional control and relyheavily on translational control strategies to modulate viralgene expression.

Translational programming, such as the use of regulatoryupstream open reading frame(s) (uORFs), overlapping read-ing frames, multicistronic transcripts, and termination control,allows viruses to conserve the functional genome size by mak-ing efficient use of genome coding capacity. In general, themechanisms of translational programming are intrinsic to thestructure of the viral mRNA itself. As summarized in Table 1,structural elements within a viral mRNA that affect transla-tional efficiency or impart translational control include thelength and structural complexity of the 59 and 39 untranslatedregions (UTR), the position and context of the initiator AUGcodon, the stability and accessibility of the of the m7G cap andthe cap-binding complex, and the presence of uORF(s) pre-ceding the major cistron (137, 148, 149, 269, 270, 322, 329, 430,431). In addition, cis-acting sequence elements that recruit orbind trans-acting factors can impart an additional level oftranslational control to viral mRNA by facilitating transla-tional selectivity (23, 76, 187, 244, 361, 362, 394, 411). Asdescribed in the following sections, virus translational pro-gramming affects all levels of the translation process, includingtranslation initiation, elongation, termination, and host trans-lational control signaling pathways.

Translation initiation. The majority of control over cellularmRNA translation occurs during initiation. Translation initia-tion is the process in which the mRNA assembles into a mac-romolecular complex with the components required for pro-tein synthesis, including the eukaryotic initiation factors (eIF)

and elongation factors (EF). Figure 2 shows the major steps inthe cap-dependent translation initiation process and importantsites of virus regulation (for comprehensive reviews of trans-lation initiation, the reader is referred to references 163, 221,335, and 359). Initiation begins with the binding of initiatormethionyl-tRNA (Met-tRNAi) to the 40S ribosomal subunit.This step is facilitated through the formation of an eIF2–GTP–Met-tRNAi ternary complex (462). The recent discovery andfunctional analyses of eukaryotic homologues of prokaryoticinitiation factor 2 (IF2) indicates that Met-tRNAi delivery alsoproceeds via a more general, universally conserved mechanism(60, 291). In this case, IF2 does not participate in the formationof a ternary complex but, rather, may bind directly to theribosomal A site and facilitate binding of the Met-tRNAi to theribosomal P site during translation initiation. IF2 activity is notsubject to direct regulation and therefore may not contributeto the control of mRNA translation. In contrast, formation ofthe eIF2-dependent ternary complex and its delivery of Met-tRNAi to the 40S ribosomal subunit can constitute a rate-limiting step when the alpha subunit of eIF2 (eIF2a) is phos-phorylated by specific protein kinases (see below) (65, 335).Phosphorylation of eIF2a thus represents a major point ofcontrol over the translation initiation process. eIF2a phosphor-ylation dramatically alters the efficiency and rate of mRNAtranslation and is a critical component of antiviral and cellgrowth control pathways (243, 321, 322, 335). eIF2 directs theternary complex to the 40S ribosomal subunit to form a 43Spre-initiation complex that includes eIF3 (Fig. 2). eIF3 facili-tates binding of the 43S pre-initiation complex to the mRNAvia the cap-binding complex, eIF4F, that has been assembledaround the mRNA m7G cap structure (335, 359).

Cap-binding reaction. Assembly of the eIF4F complex onthe mRNA is dependent on the eIF4E component of thiscomplex, which recognizes and binds the m7G cap (173). Theaffinity of eIF4E for the m7G cap constitutes a second majorcontrol point in the translation initiation pathway and is sub-ject to variation through eIF4E phosphorylation (237, 315,433). In addition, the cap-binding activity of eIF4E can beblocked through the formation of an eIF4E-eIF4E bindingprotein (4E-BP) complex, resulting in inhibition of cap-depen-dent translation (367, 433). Formation of the eIF4E/4E-BPcomplex itself is subject to regulation through 4E-BP phos-phorylation and dramatically affects cell growth control byaltering the efficiency and selectivity of mRNA translation (re-viewed by Sonenberg and Gingras [433]). As discussed in detail

TABLE 1. mRNA structural features that confer translational control.

Structural feature Effect on translation References

Length of 59 UTR Influences scanning. Long 59 UTR may impede initiation. 11, 107, 432Secondary structure of 59 UTR

and coding regionComplex 59 UTR structures may impede scanning. IRES structures promote cap indepen-

dence and allow the ribosome to largely avoid scanning. Pseudoknot structures promoteframeshifting and recoding. May mediate binding with trans-acting factors.

12, 23, 107, 118, 221,223, 286, 396, 430

Sequence context of theinitiation codon

Imparts ribosome selectivity for first AUG codon. “Weak” AUG codon promotes leakyscanning.

221, 271, 272

M7G cap Promotes mRNA stability and interaction with eIF4F. Facilitates the translation of mostcellular mRNAs. Accessibility to initiation factors may influence translational efficiency.

17, 136, 300, 335, 432,442, 457

uORF May impede ribosome scanning to downstream cistron(s). Multiple uORFs promote theselective translation of GCN4 in yeast.

146, 148, 202, 342

Poly(A) tail Length imparts stability and translational efficiency to mRNA. Interaction with PABP medi-ates association with cap-binding complex on the mRNA.

27, 136, 225, 289, 393,401

39 UTR Mediates closed-loop translation complex via PABP interaction. Structural complexity mayinfluence translational efficiency and interaction with trans-acting factors.

63, 75, 225, 276, 401,430, 452, 466, 479,492

Codon usage Use of nonabundant tRNAs may impede elongation. Influences frameshifting and recoding. 12, 40, 152trans-acting factors Specific RNA sequence and/or structural motifs promote interaction with RNA-binding

proteins, which may influence translational efficiency.63, 335, 426, 492

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below, these regulatory steps are targeted by a group of viruses,which are best defined by the family of picornaviruses andincludes poliovirus and encephalomyocarditis virus (EMCV)(155, 174, 322). These viruses initiate translation through acap-independent mechanism that involves internal ribosomeentry through use of the internal ribosome entry site (IRES);virus-mediated cleavage of the 220-kDa cap-binding protein,eIF4G (161, 174, 283); and dephosphorylation of the 4E-BPsto sequester eIF4E in an inactive eIF4E/4E-BP complex (Fig.2) (155). IRES-mediated translation requires specific cis-actingsequences within the viral RNA that mediate the interac-tion with trans-acting host factors. Thus, the global process ofIRES-mediated translation essentially eliminates the competi-tion for host factors from cap-dependent cellular mRNA trans-lation, favoring the translation of viral mRNA.

Ribosome scanning and AUG site selection. Following asso-ciation with the mRNA, the 43S preinitiation complex beginsscanning from the 59 end of the mRNA or the site of ribosomeentry (as in the case of cap-independent translation) and con-tinues scanning until the Met-tRNAi interacts with the initiatorAUG codon. Ribosomal scanning is not always compatiblewith mRNAs that possess a long and/or structured 59 UTR. Asdescribed in “Mechanism and control of viral mRNA transla-tion” (below), viral mechanisms to overcome the inefficiency of

scanning the 59 UTR and to bypass the host shutoff phenom-enon include the use of internal ribosomal entry on the mRNAand the ribosomal shunt (93, 223, 494). These mechanismsallow the preinitiation complex to effectively avoid a large partof the 59 UTR and begin scanning within the region of theinitiator AUG codon on the viral mRNA (Fig. 2).

Once the Met-tRNAi associates with the initiator AUGcodon, GTP is hydrolyzed from the ternary complex, boundinitiation factors are released, and the 60S ribosomal subunitjoins the preinitiation complex. The resulting 80S initiationcomplex then mediates the elongation phase of translation. Inthis model, initiation begins at the 59-proximal AUG codon(221, 335). However, the AUG site selection for translationinitiation is dependent in part on the context of the AUGcodon, where the canonical accAUGg sequence (initiationcodon in capitals) exerts the highest preference for initiation(221, 270). Departure from this sequence is associated withleaky scanning, in which the preinitiation complex will recog-nize a noncanonical or weak AUG only at a low frequency andscans past to initiate translation at a downstream codon moreclosely matching the canonical initiator AUG (Fig. 2) (221).Leaky-scanning initiation of translation is popular among vi-ruses, and in retroviruses it can provide a mechanism for

FIG. 2. Schematic illustration of eukaryotic mRNA translation and major sites of viral regulation. Details of the translation process are described in the text.Translation initiation factors are shown by their letter and number designation (335). 40S and 80S denote the small ribosomal subunit and the elongating ribosome,respectively. 1, Ternary-complex formation and assembly of the 43S pre-initiation complex; 2, assembly of the cap-binding complex and ribosome loading onto themRNA; 3, Ribosome scanning to the first AUG codon, recycling of eIF2-GDP, and joining of the 60S ribosomal subunit. TER denotes a translation termination codon.Major sites for viral control of translation and mechanisms of translation control are shown in the surrounding boxes. Not shown is the mRNA 39 UTR, which can alsoinfluence translational efficiency. aa, amino acid.



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achieving defined stoichiometric ratios of translation products(412, 413).

Elongation. During the elongation phase of translation, themRNA is associated with multiple 80S ribosomes, or polyribo-somes, as amino acid residues are sequentially placed on thecarboxyl end of the growing peptide chain. In many virus sys-tems the replicative cycle is demarked by early- and late-stageevents that can be distinguished by the differential recruitmentof viral mRNA into polyribosomal complexes at specific timesafter infection. As with herpes simplex virus type 1 (HSV-1),this often coincides with the synthesis of latency factors anddeterminants of virulence (114, 159, 279). The process of trans-lation elongation itself is subject to viral regulatory control(Fig. 2). Elongation control mechanisms include ribosomalframeshifting (108), functional recoding (151), and virus-direct-ed modification of EF-1 (257); the first of these is prevalent inretroviruses and reveals otherwise cryptic ORFs within theviral mRNA (78, 108, 109, 151).

Termination. The process of translation termination occurswhen the translating 80S ribosome encounters an in-frametermination codon within the template mRNA. The termina-tion codon is recognized by a release factor, which mediatesthe hydrolysis of the peptide chain from the bound tRNA (335,501). This results in the release of the nascent polypeptidefrom the 80S ribosome and leads to the eventual dissociationof the ribosomal subunits. Once termination has occurred, the40S subunit is free to continue scanning the mRNA (Fig. 2). Inmulticistronic transcripts, termination can be followed by reini-tiation at the downstream cistron, subject to ternary-complexavailability (221). However, reinitiation is usually very ineffi-cient, and the presence of a uORF can confer limitations to thetranslational efficiency of the major, downstream ORF. Asdescribed in “Frameshifting” (below), this termination-re-initiation translational control mechanism is prevalent amongviruses and is used to control the synthesis of specific viral geneproducts (148).

Improving translation efficiency: the closed-loop model ofmRNA translation. Since the discovery of 39 polyadenylation ineukaryotic mRNA, it has become quite clear that the poly(A)tail imparts stimulation of mRNA translation in eukaryotes(reviewed by Jacobson [225]). More recent analyses indicatedthat the translation-stimulatory function of the poly(A) tail wasdue, in part, to the actions of the poly(A)-binding protein(PABP). In mammalian cells, PABP interacts with elements ofthe cap-binding complex assembled on the 59 end of themRNA, thus rendering a “closed-loop” translation complex(Fig. 3) (138, 225, 401). PABP promotes the closed loop by

binding to eIF4G and to PABP-interacting protein 1 (Paip-1)(75). Paip-1 interacts with components of the mRNA cap-binding complex, including eIF4G and the eIF4A helicase(306). Analyses of translation initiation in yeast and plantsindicate that the interaction between PABP and eIF4G stim-ulates mRNA translation (289, 452, 476). The proximity of themRNA ends provided by the closed-loop translation complexis thought to contribute to the stability of the mRNA and the59 cap complex and to provide for the efficient recruitment andrecycling of ribosomal subunits (225). Thus, the overall effectof the closed loop is to increase translation efficiency. Virusesexploit the closed-loop translation complex as a means of re-directing the host translation machinery to favor viral mRNAtranslation. As described below, viruses accomplish this bytargeting PABP and disrupting the interaction of the mRNAends, resulting in attenuation of host mRNA translation (233,259, 376).

TRANSLATIONAL CONTROL OF VIRALGENE EXPRESSION

Translational dependence has driven viruses to adopt trans-lational programming that maximizes efficiency and facilitatesthe selective translation of viral mRNA over the endogenoushost transcripts. Viral translation strategies have evolved toutilize both the advantages and the limitations inherent withinthe cap-dependent host translation process. These range fromcap-dependent translation competition strategies to cap-inde-pendent strategies of IRES-mediated translation initiation. Asdiscussed below, such strategies allow viral mRNA translationto persist, even under the extreme conditions imposed by thehost shutoff phenomenon, which severely limits cellular me-tabolism. This section describes the various translation strate-gies utilized by eukaryotic viruses to overcome the problemsassociated with translational dependence and concludes with adiscussion of how viral translational programming may presentnovel targets for the development of anti-viral therapeutics.

Advantages and liabilities of cap-dependent host transla-tion. The majority of mRNA translation within eukaryotic cellsis dependent on the m7G cap, a unique structure present at the59 terminus of the mRNA (335). The 59 cap promotes mRNAstability and nuclear export and provides for various levels ofcontrol over the translation initiation process. Cap-dependentcontrol of mRNA translation confers several advantages to thecell. First, and perhaps most importantly, cap dependency al-lows the cell an immediate mechanism through which to con-trol gene expression by modulating the assembly and activity ofcap-binding complex components. Second, cap-dependencyprovides selectivity of translation by combining the transla-tional regulatory properties inherent within a specific mRNAwith those due to modification of the cap-binding complex.Translational control thereby allows the cell to fine-tune geneexpression by stimulating or repressing the translation of spe-cific mRNAs, usually through the reversible phosphorylationof translation factors (335).

While cap-dependent translation clearly affords several ad-vantages to the host cell, it also presents liabilities that areeffectively exploited by viruses. Cap dependency requires anintact pool of specific initiation factors, namely, the compo-nents of the eIF4F cap-binding complex (100, 292, 335, 457,468). Moreover, it necessitates the capping and nuclear exportof mRNAs. Viruses have learned to disrupt these processes inorder to reprogram the host cell toward the synthesis of viralproteins. Viral disruption of cap-dependent host translationcontributes to the host shutoff that is often observed duringproductive infections (10).

FIG. 3. Model of the closed-loop mRNA translation complex. The mRNA-bound eIF4F initiation complex interacts with the 39 end of the mRNA viaPABP. Poly(A) sequences within the 39 UTR direct PABP binding to themRNA. PABP mediates interaction with the cap-binding complex either directlythrough eIF4G (4G) (452) or indirectly through an eIF4G, eIF4A (4A)-depen-dent interaction with Paip-1 (75). Assembly of the closed-loop complex maystabilize the interaction of the 40S ribosomal subunit with the mRNA (225, 401).



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Host Shutoff and Selective Translation of Viral mRNA

Host shutoff is the process in which cellular macromolecularsynthesis is suppressed due to viral domination of host metab-olism that occurs during infection (reviewed in reference 10).Host shutoff is not an absolute; not all virus infections exhibithost shutoff, and shutoff is not always required to facilitate viralreplication. Within the many viral systems in which host shutoffis known to occur, shutoff ultimately favors the translation ofviral mRNA over endogenous host transcripts, although hostshutoff itself may not be directly attributed to viral disruptionof host mRNA translation (1, 10). The selective translation ofviral mRNA during the host shutoff is clearly a multicompo-nent process that has been attributed to a variety of factors.These include viral perturbation of intracellular ion concen-tration (144) and nucleotide metabolism (215, 244, 279), alter-ations in RNA stability, processing, and export (119, 245, 311,352, 497), and the recruitment of specific host factors (249,294). From a simpler perspective, the selective translation ofviral mRNA during host shutoff may reflect a general compe-tition between viral and host mRNA for the translational ma-chinery. For example, host shutoff in cells infected with vesic-ular stomatitis virus (VSV) is coupled to the selectivetranslation of viral mRNA (Fig. 4, lanes 3 and 4). Interestingly,however, the abundance and stability of cellular mRNAs andtheir efficiency of translation initiation remain unaltered (54).Examination of VSV-infected cells revealed that the preferen-tial translation of viral mRNAs was a result of ribosome com-petition from an overwhelming abundance of viral mRNA(309). At the other end of the spectrum is the host shutoff thatoccurs during picornavirus infection. In this case, the shutoff ofhost protein synthesis and selectivity for viral mRNA transla-tion is clearly a virus-directed event mediated, in part, throughcleavage of eIF4G by the virus-encoded 2A protease (2A-pro)(174, 273, 468). Cleavage of eIF4G by 2A-pro disrupts cap-

dependent translation initiation to favor the IRES-mediatedtranslation of the picornavirus mRNA (Fig. 4; also see Fig. 14)(23). Similarly, the host shutoff in cells infected with influenzavirus features a strong selection for viral mRNA translation(Fig. 4, lanes 5 and 6). However, unlike the picornaviruses,influenza virus mRNA translation is cap dependent (251). Inthis case, the predominance of viral protein synthesis is facili-tated, in part, by virus-mediated endonucleolytic cleavage ofthe host mRNA m7G cap, subsequent mRNA destabilization,and the dephosphorylation of eIF4E (244). The selectivity ofviral mRNA translation is then mediated through the recruit-ment of the cellular G-rich sequence factor 1 (GRSF-1) pro-tein and other host factors to the 59 leader sequence of theinfluenza virus mRNAs (361, 362).

Host shutoff can be seen as beneficial for viruses, since itplaces cellular resources largely at their disposal. However, theshutoff phenomenon also presents several challenges to thevirus, not least of which is maintaining the integrity of the hostcell long enough to complete the virus replicative cycle. This isespecially important from the standpoints of translational de-pendence and viral persistence, in which the virus must ensurethat the host translation machinery remains competent for thesynthesis of viral proteins. Problematically, however, the met-abolic repression and stress of host shutoff are potent inducersof cellular apoptosis and translational control programs thatfunction within the cellular antiviral response to block viralinfection (179). Viruses have taken a two-pronged approach tothese problems of host shutoff, and they encode mechanisms to(i) disrupt host apoptotic programs (353) and (ii) control theantiviral translational response imposed through the phosphor-ylation of eIF2a (65, 66). As described in detail in “Viralmodification of cellular factors” (below), eIF2a phosphoryla-tion by the cellular serine/threonine protein kinase (PKR) pre-sents a translational blockade to viral replication (68, 131).Disruption of host apoptosis and the phosphorylation of eIF2atherefore facilitates viral replication by maintaining host cellintegrity and ensuring translational competence during hostshutoff.

The relationship between translational control, apoptosis,and viral infection has been an intense area of study in recentyears. The emerging picture now suggests that translationalsuppression through eIF2a phosphorylation is an importantcomponent of apoptotic programming (450). Thus, disruptionof eIF2a phosphorylation may serve the dual purpose of main-taining the translational competence of the host cell and pre-venting apoptosis during host shutoff. This idea is supported bythe many studies of vaccinia virus replication in which the viralK3L and E3L gene products have been implicated in disrupt-ing eIF2a phosphorylation and blocking apoptosis (52, 55, 82,83, 135, 255, 420). Moreover, studies by Roizman and col-leagues have demonstrated that disruption of eIF2a phosphor-ylation by the HSV-1 g134.5 gene product was a requisite forsustained translational competence and viral persistence dur-ing the host shutoff induced by HSV-1 infection (188, 190).Influenza virus similarly ensures that eIF2a phosphorylation isblocked and translational competence is maintained duringhost shutoff (244, 294). However, rather than preventing shut-off-induced apoptosis, influenza virus may delay or reprogramapoptosis to facilitate cell lysis and virion release during late-stage infection (116, 445, 446). In closing this section, it isimportant to note that maintenance of translational compe-tence during host shutoff may ultimately contribute of viralpathogenesis. As described in “Viral persistence and transla-tional control” (below), the ability to suppress mRNA trans-lation is a key component for the control of cell growth. Inpersistent viral infections, such as those by hepatitis C virus

FIG. 4. Virus-induced shutoff of host cell protein synthesis. Murine NIH 3T3cells (lanes 1 to 4) or Madin-Darby bovine kidney cells (lanes 5 and 6) were mockinfected (U) or infected (I), respectively, with EMCV (lanes 1 and 2), VSV(lanes 3 and 4), or influenza virus (lanes 5 and 6). To visualize the virus-inducedhost shutoff of protein synthesis and the concomitant shift to viral proteinsynthesis, proteins were biosynthetically labeled by the addition of [35S]methi-onine to the culture medium. Protein equivalents from mock-infected and virus-infected cells were separated by gel electrophoresis and visualized by autora-diography of the dried gel. Arrows denote the positions of viral proteins. Thepositions of molecular mass standards are indicated in kilodaltons.



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(HCV) or the DNA tumor viruses, constitutive modulation ofhost translational control pathways and release of translationalsuppression may make important contributions to viral onco-genesis (125, 134, 248, 444).

Mechanisms and Control of Viral mRNA Translation

Viruses utilize the canonical translation factors and machin-ery of the host cell to facilitate completion of their transla-tional programming. Figure 5 depicts various means by whichviruses implement their translational programming toward thecommon end of synthesizing viral proteins and completing thevirus life cycle. Reflecting the nature of the virus-host rela-tionship itself, the host cell has evolved countermeasuresthat impose blockades upon viral protein synthesis. As de-scribed below, viral translational programming often includesmechanisms to manipulate the host translational machineryand overcome these antiviral blockades.

Internal ribosome entry. Translation initiation, mediatedthrough the internal entry of ribosomes onto the substratemRNA, was first found in 1988 during studies of poliovirus andEMCV replication (227, 370). Examination of the nucleotidesequence of the picornavirus 59 UTR has revealed a region ofsignificant secondary structure spanning approximately 500 nu-cleotides (nt) and punctuated by multiple AUG codons (222,228). This region was initially known as the ribosome landingpad and later termed the internal ribosome entry site (IRES)(for detailed reviews of the IRES, see references 193, 223, and329). Translation studies performed in vitro and in vivo dem-onstrated that the IRES could confer internal ribosome entryto a downstream ORF when placed between the cistrons of amulticistronic mRNA (226, 227, 370). Moreover, incorporationof the IRES to precede the ORF of a circular mRNA facili-tated ribosome entry and translation of the circular cistron(57). These studies concluded that the IRES is a genetic ele-

ment that facilitates internal ribosome entry and mRNA trans-lation independent of the m7cap structure. Since then, theobservation of IRES-mediated translation has been extendedto include other virus families, most notably the other picor-naviruses and the members of the genera Pestivirus and Hepa-civirus of the family Flaviviridae (which include bovine diarrheavirus and HCV, respectively [Table 2]) (223). We also note thatIRES-mediated translation of certain cellular mRNAs has alsobeen identified and may constitute a minor proportion of totalcellular mRNA translation within the cell (223, 234, 235, 347).

Picornaviruses, pestiviruses, and hepaciviruses carry onecopy of an uncapped, single-stranded RNA of positive polaritythat functions directly as the viral mRNA and substrate fortranslation (117). These virus families translate their genomicRNA as a single large polyprotein that is posttranslationallyprocessed into distinct structural and nonstructural polypep-

FIG. 5. Viral mechanisms of translational programming. The top diagram shows structural features of a representative mRNA containing a m7G cap and consistingof a series of overlapping and nonoverlapping reading frames (denoted by rectangles). The first reading frame is indicated by an AUG initiation codon and is precededby a 59 UTR. Upright arrows indicate translation initiation of the corresponding reading frame(s), resulting from the mechanisms listed at left. Specific viruses examplespresented within the text are listed at right. Depiction of the IRES and ribosome shunt includes the relevant stem-loop structures within the 59 UTR. Arrow showsribosome bypass, or shunting, around the stem-loop. Figure adapted with modification from reference 322.

TABLE 2. Representative viruses that utilizeIRES-mediated translation

Group Representative members

Enteroviruses and rhinovirusesa

Human rhinoviruses..........................Common cold virus/many serotypesEnteroviruses .....................................Poliovirus, coxsackieviruses, entero-

virus 70, echoviruses

Cardioviruses and aphthovirusesa

Cardioviruses .....................................Encephalomyocarditis virus, mengo-virus

Aphthoviruses ....................................Foot-and-mouth disease virus

Hepatovirusesa .......................................HAVPestiviruses .........................................Bovine diarrhea virusHepaciviruses .....................................HCV

a Groupings are based on picornavirus IRES sequence homologies (42, 192,483).



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tide products through a series of proteolytic cleavage events(for reviews of the replication of picornaviruses, pestiviruses,and hepaciviruses, see references 23, 33, 117, 390, and 398).Picornavirus infections generally exhibit an intense host shutoff(with the exception of hepatitis A virus [HAV]) that is char-acterized by IRES-mediated selective translation of the viralmRNA (Fig. 4, lanes 1 and 2) and rapid lysis of the host cellwithin 6 to 12 h after infection (398). In contrast, the prototypichepacivirus, HCV, mediates persistent infection in which theeffects, if any, on host shutoff are less well understood. What,then, is the role of the IRES element in the contrasting lifecycles of these two virus families?

The functional role of the IRES is perhaps best understoodby considering IRES structure, the context of the IRES withinthe 59 UTR, and the advantages conferred by IRES-mediatedtranslation. IRES secondary structure has been concisely mod-eled from several different viruses, and the prototypic IRESstructures in poliovirus, EMCV, HAV, and HCV are depictedin Fig. 6. The extensive secondary structure of the IRES makesthe long viral 59 UTR (which ranges from 341 nt in HCV toover 1,400 nt in various picornaviruses) incompatible withstandard 59 ribosome entry, scanning, and AUG site selection.Among picornaviruses, the IRES itself spans approximately450 nt and begins a variable distance from 59 terminus of theRNA (101). The enteroviruses and rhinoviruses comprise astructurally conserved IRES group that is distinct from a sec-ond IRES group represented by the cardioviruses and aphtho-viruses (193, 223). A third and structurally distinct picornavirusIRES group is represented by HAV (156).

The IRES groups also diverge in relation to the position ofthe authentic initiator AUG codon. Both the cardiovirus-aph-thovirus IRES group and HAV initiate translation from theAUG codon at the immediate 39 boundary of the IRES (193).Thus, the site of ribosome entry actually corresponds to theinitiation codon. In contrast, the enteroviruses and rhinovi-ruses initiate translation from an AUG codon located approx-imately 40 and 160 nt downstream, respectively, from the 39

IRES boundary (23, 223). In this case, the ribosome scans themRNA from the point of ribosome entry to the authenticAUG codon, in accordance with the conventional scanningmodel. The prototypic HCV IRES diverges in both length andstructure from the three picornavirus IRES groups (Fig. 6).The actual boundaries of the HCV IRES have yet to be pre-cisely defined, but initial studies suggest that the IRES beginsapproximately 20 nt from the RNA 59 terminus and extends atleast 30 to 40 nt into the actual coding region of the viral coreprotein (211, 391). Ribosome entry on the HCV RNA takesplace within the IRES itself rather than at the 39 IRES bound-ary. This coincides with the actual site of translation initiation,which resides approximately 40 nt internal to the 39 IRESboundary (391).

Structural conservation between picornavirus IRES groupsis limited to a 10- to 15-nt pyrimidine-rich sequence elementlocated near the 39 IRES boundary (101) and, to a lesser ex-tent, a common 39 structural core related to the group I intron(290). The significance of the latter is not clear, although it mayreflect common structural features of RNA required for theassembly of mRNP complexes. Several independent studieshave identified the pyrimidine-rich sequence element as a con-served IRES feature, indicative of a role for this element inIRES-mediated translation. Indeed, partial deletion or purinesubstitution of the pyrimidine-rich sequence abolished thefunction of the poliovirus IRES and, to a lesser extent, limitedthe translation efficiency of the EMCV IRES (192, 351). To-gether, these experiments demonstrated that the number ofbases residing between the 39 end of the pyrimidine-rich se-quence and the actual initiator AUG codon was an importantvariable influencing the site of ribosome entry and AUG codonselection. These results suggested that (i) the length of thepyrimidine-rich sequence element and its proximity to the ini-tiator AUG codon may influence translation start site selectionand (ii) this sequence element may function in the process ofIRES-mediated recruitment of the translation initiation com-plex to the site of initiation. The latter notion is supported by

FIG. 6. IRES structure. Structural representation of the 59 UTR from EMCV (top left), poliovirus (top right), HAV (bottom left), and HCV (bottom right). Themajor stem-loops are labeled according to previous designations (42, 193, 206, 483). The region encompassing the IRES is underlined. Pyrimidine-rich sequence el-ements are shown as solid rectangles. AUG denotes the position of the translation initiation codon. The box on the HCV IRES denotes the core protein-coding region.



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the findings that several host proteins, including the cellularpoly(rC)-binding protein 2 (PCBP2) and the pyrimidine tract-binding protein (PTB), may interact with the pyrimidine-richsequence element and affect the translation efficiency of thepicornaviral IRES (34, 35, 238, 264, 394).

Interestingly, the HCV genome contains a pyrimidine-richsequence element located just outside the IRES, at the 39 endof the core-coding region (206) (Fig. 6). Recent analyses indi-cate that the HCV core region pyrimidine-rich sequence func-tions in cis to regulate translation from the HCV IRES. Inthese studies, Ito and Lai found that the core region pyrimi-dine-rich sequence had a suppressive effect on IRES-mediatedtranslation, most likely induced through interaction with PTB(217, 218). This translational suppression was relieved by aninteraction of PTB with the highly conserved 98 nt of the HCV39 UTR. These results are consistent with the idea that se-quences outside the HCV IRES impart control over the trans-lation initiation process and that this may involve (i) the re-cruitment of a host factor(s) to the HCV RNA, and (ii) crosstalk between the viral 39 UTR, internal elements in the HCVRNA, and the IRES itself. Such a model may provide a mech-anism allowing the virus to modulate polyprotein synthesis inresponse to changing conditions within the host cell throughspecific protein interactions with the viral RNA.

IRES-mediated translation avoids the potential limitationsposed by cap dependency and provides important advantagesfor viral replication. By mediating internal ribosome entry, theIRES allows the viral mRNA to bypass rate limitations ontranslation imposed by the cap-binding reaction. This can beseen as critical for the picornaviruses, which can reach anastounding rate of 5 3 106 virion particles/cell over a 6-hperiod (398), and HCV (1012 virion particles/day/ml of infectedblood examined [348]). Cap-independent translation bypassesthe requirement for a virion-encoded mRNA-capping enzymeor alleviates the requirement for host capping enzymes andnuclear localization of viral mRNA synthesis and replication.The picornaviruses have taken this a step further by encodingmechanisms to disrupt cap-dependent translation in favor ofviral protein synthesis. As described in “Viral modification ofcellular factors” (below), these involve cleaving the eIF4Gcomponent of the cap-binding complex (174, 468), regulatingthe activity of eIF4E (155), and cleaving PABP (233, 259).Perhaps most importantly, the IRES allows the 43S preinitia-tion complex to avoid scanning the long 59 UTR of the viralmRNA. Thus, IRES entry of ribosomes avoids the pitfalls ofscanning through highly structured regions within the viralmRNA and bypasses any initiation interference from upstreamAUG codons and uORFs (Fig. 5). This also ensures that thetranslation machinery avoids interfering with the genome rep-lication signals embedded within the 59 UTR of the viral RNA(37).

As suggested by the IRES-mediated translation model(223), IRES function is determined, in part, through interac-tions with host proteins in addition to the translational ma-chinery itself. IRES-host protein interactions may contributeto host range specificity and virulence phenotype. This wassuggested by early experiments that measured the in vitrotranslation efficiencies of the poliovirus and EMCV IRES el-ements within a rabbit reticulocyte lysate translation system(41, 99). These experiments found that the cardiovirus andaphthovirus RNAs were efficiently translated in vitro whereas,in contrast, the enterovirus and rhinovirus RNAs were trans-lated with low fidelity and inefficiency. Supplementation of thetranslation mixture with HeLa cell extract significantly in-creased IRES efficiency and restored the accuracy of transla-tion (371). These studies immediately suggested that viral host

range might directly reflect the requirements for specific hostfactors, in addition to the canonical translation factors, tofacilitate IRES function and viral protein synthesis. Indeed,IRES-binding host factors have now been identified that play afunctional role in IRES-mediated translation (23, 372, 394)(see below). The identification of such factors, and their cog-nate binding sites within the IRES, should lead to a betterunderstanding of the impact of translational control on hostrange restriction and viral pathogenesis.

The concept that the translational efficiency of viral mRNAaffects pathogenesis is supported by the results of analyses ofpoliovirus Sabin vaccine strains (400). A subset of attenuatingmutations within the Sabin strains were mapped to the majorstem-loop V (Fig. 6) of the poliovirus IRES (reviewed byEhrenfeld [101]). These mutations resulted in reduced trans-lation efficiency of the viral mRNA, resulting in the attenuatedvaccine phenotype. These studies suggested that the stem-loopstructures within the IRES were critical determinants of trans-lational efficiency and, in poliovirus, were important elementsof neurovirulence. Analyses of the neuropathogenic phenotypeof rhinovirus/poliovirus chimeras support this notion. Studiesof poliovirus neurovirulence in which the poliovirus IRES wasreplaced with the structurally related IRES from human rhi-novirus type 2, conducted by Wimmer and colleagues (167),demonstrated that the neurovirulent phenotype of polioviruswas dependent on the authentic poliovirus IRES. Interestingly,the chimeric virus retained its ability to replicate in nonneu-ronal tissues, indicating that the IRES itself is an importantdeterminant of host range specificity. More recent structure-function analyses have identified stem-loops V and VI as thepoliovirus genetic elements responsible for the neurovirulentphenotype (168). Together, these results suggest that poliovi-rus IRES stem-loops V and VI provide the capacity for viralreplication within the central nervous system. It is thereforepossible that these genetic determinants may function to re-cruit tissue-specific host factors to the poliovirus IRES. Futureexperiments to test this idea should aid in our understanding ofthe relationship between IRES structure and viral pathogene-sis.

Substantial progress is now being made toward understand-ing how elements within the HCV IRES may affect viral rep-lication and pathogenesis. An initial comparison of IRESstructure and IRES-dependent translation from representativeisolates of the various HCV genotypes has revealed some in-teresting features. First, by measuring the relative translationrates of chloramphenicol acetyltransferase (CAT) reporterconstructs placed under control of HCV IRES sequences, Bu-ratti et al. demonstrated a lower translation efficiency for HCVgenotype 3 compared to genotypes 1 and 2 (47). Moreover,these investigators found that conservation of both the second-ary structure and the sequence within linear domains of HCVIRES stem-loop III was important for maintaining IRES func-tion. A broader analyses of HCV IRES translational efficiencysupported and extended these results, revealing a lower effi-ciency of translation mediated by the IRES elements of HCVgenotypes 3 to 6 (71). It is interesting that the level of poly-morphism between the IRES elements used in the latter studywas limited to a mere total 17 nucleotide positions. Most ofthese differences could be mapped to within the regions on themajor stem-loop III and, to a lesser extent, stem-loop IV. Thus,it appears that subtle differences in nucleotide sequence canconfer sufficient alterations in RNA secondary structure toaffect the function of the HCV IRES and the efficiency of viralmRNA translation. In accordance with this idea, Honda et al.have revealed that major alterations in stem-loops III and IVof the HCV IRES resulted in loss of function, presumably due



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to the induction of gross aberrations in IRES structure (204–206). However, these studies also identified differences intranslation efficiencies between HCV genotypes 1A and 1Bthat were not attributed to major structural differences be-tween the respective IRES sequences (206). In follow-up stud-ies, it was found that the reduced translation efficiency of theHCV 1B RNA was attributed to the presence of an AG dinu-cleotide sequence (HCV nt 34 and 35) present within a single-stranded region that preceded the IRES (207). Mutation of theAG dinucleotide to GA (present in HCV 1A) increased thetranslation efficiency of HCV 1B RNA, demonstrating that thewild-type (wt) AG dinucleotide sequence had an inhibitoryeffect on translation. Remarkably, translation inhibition wasattributed to an RNA-RNA interaction mediated between theAG dinucleotide sequence and a region far downstream withinthe viral core coding sequence (207). These results suggest thatthe long-range RNA-RNA interaction between the viral 59UTR and the core-coding region functions cooperatively toinfluence the activity of the HCV IRES. Such an interactionmay sufficiently alter the structure of the IRES to affect theefficiency of viral RNA translation. An intriguing question iswhether the correlation between HCV genotype and IREStranslation efficiency in vitro extends to the phenotypic differ-ences exhibited by these viral genotypes during the course ofHCV infection.

Molecular epidemiological studies have identified an asso-ciation between HCV genotype, response to the current anti-HCV interferon (IFN) therapy, and severity of infection. Inthese studies, patients who were infected with HCV genotype1 or 2 consistently presented a more severe pathology thandid patients infected with HCV genotype 3, 4, 5, or 6 (7, 45).Moreover, analyses of the virus load in serum revealed anassociation between increased viral titer, resistance to therapy,and a poor prognosis, especially in patient groups infected withHCV genotype 1 (103, 115). It has been proposed that thesebiological differences between HCV genotypes may be due, inpart, to the variations in translation efficiency conferred by thesubtle polymorphisms between the respective genotype-spe-cific IRES. Secondary-structure analyses of the HCV IRESisolated from healthy patients with low viral titers who re-sponded to anti-HCV therapy and from those who did notrespond and maintained high viral loads revealed that a sig-nificant level of IRES structural variation was associated witha low viral titer and complete response to therapy (439). Instark contrast, conservation of IRES structure was consistentlyassociated with maintenance of a high virus load and resistanceto therapy. Taken together, these studies suggest that IRESstructure and the corresponding translation efficiency of agiven HCV strain are critical factors affecting the virulence ofHCV and the sensitivity to antiviral therapy. Such results haveidentified the HCV IRES as a potential target for therapy ofHCV infection (31).

Ribosome shunt. The processes of cap-mediated ribosomeentry, 59 scanning, and internal initiation are combined fea-tures of the ribosome shunt mechanism of translation. Ribo-some shunting allows the small ribosomal subunit to avoid theproblems of scanning a long and complex linear sequencepreceding the major ORF. Unlike IRES-mediated translation,shunting is dependent on the 59 cap and the cap-binding com-plex for ribosome entry onto the mRNA (410) (Fig. 5). Thebest evidence for ribosome shunting, and the general ribosomeshunt model, comes from studies of the pararetrovirus cauli-flower mosaic virus (CaMV) (94, 410), although shunting alsotakes place in other viral systems (reviewed in reference 203).Upon engaging the mRNA, the ribosome scans until it reachesa cis-acting shunting element that promotes ribosomal trans-

location to a downstream receiving element(s) (94). By thisprocess, scanning becomes nonlinear and the ribosome canbypass a large portion of the 59 UTR to initiate translation atthe downstream cistron (Fig. 5 and 7).

CaMV encodes two major RNAs, a monocistronic 19S RNAthat encodes a translational transactivator and the 35S prege-nomic RNA (123). The 35S RNA spans the complete CaMVgenome, serves as the template for virus-mediated reversetranscription, and functions as the major viral mRNA for thesynthesis of at least seven viral proteins. Translation from the35S RNA is cap dependent and is under the control of the600-nt multifunctional RNA leader (196). Structural analysesof the CaMV leader revealed that it contains several shortuORFs and assumes a complex stem-loop structure (123, 195).Although these features of the CaMV leader are incompatiblewith the 59-scanning model, translation from downstreamORFs still occurs with reasonable efficiency in vitro and in vivo(410).

Previous studies of CaMV translation demonstrated thatribosome shunting could occur in trans using separate RNAmolecules (124). These experiments involved RNAs that weredesigned to restore the secondary structure of the 59 leader byannealing and adopting the native structure of the 35S RNA.The results identified the 35S 59-UTR stem-loop structure as arequisite element in the ribosome shunting process and impli-cated the immediate flanking sequences as the shunt donorand shunt acceptor sites. Subsequent structure-function anal-yses of the CaMV stem-loop demonstrated that translationalcontrol of the CaMV 35S RNA was dependent on the presenceof an elongated hairpin comprising nt 70 to 550 of the 59 leader(195, 196). Moreover, experiments examining the influence ofthe 35S uORFs (sORFA to sORFF) on CaMV translationfound that the integrity of sORFA was required for translationof the major downstream cistrons (196). sORFA is the 59-proximal 35S uORF and is followed closely by the elongatedhairpin structure (123). These studies demonstrated that mu-tation of the sORFA initiation codon impaired translation pastthe elongated hairpin on the 35S RNA. Together, these resultsare consistent with the model that CaMV translation initiatesat sORFA, allowing assembly of the 80S ribosomal complexonto the 35S RNA (Fig. 7). Perhaps concomitantly while en-countering the termination codon of sORFA, the 80S ribo-somal complex encounters the shunt donor element at the baseof the elongated hairpin structure. sORFA translation thenterminates, with the shunt donor accepting the 40S subunit andpromoting a shunt around the elongated hairpin to the site ofthe shunt acceptor. Shunting is then followed by resumption ofmRNA scanning by the 40S ribosomal subunit. The 80S ribo-

FIG. 7. The CaMV ribosome shunt. Specific details are described in the text.The relative positions of sORFA, the major stem-loop, and the gene VII ORF ofthe 35S RNA are shown. Ribosome shunting (denoted by the lower arrow)occurs around the major stem-loop encompassing nt 70 to 550 (196). Afterterminating sORFA translation, the 40S ribosomal subunit is shunted around themajor stem-loop structure and resumes scanning to initiate translation at theORF VII AUG codon (upper arrow).



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somal complex then reassembles at the downstream ORFVIIAUG codon, and CaMV translation begins. Alternatively, theshunt donor may facilitate passage of the intact 80S ribosomalcomplex around the elongated hairpin structure without arequirement for complex disassembly, although this wouldrequire the shunt donor to deposit the ribosomal complexdirectly at the ORFVII initiation codon (Fig. 7). The exactsequence elements responsible for ribosome shunting inCaMV are not yet known. Similar to the IRES mechanism oftranslation, shunting may involve assembly of host factors atand round the shunt donor and shunt acceptor sites. Recentevidence indicates that shunting, at least in CaMV and relatedviruses, may be host independent (410). Thus, the ribosomeshunt may represent a general mechanism of translationaladaptability among viruses. Future studies to determine thenature of the host “shunting factors” will certainly aid in un-derstanding the molecular mechanisms of ribosomal shunting.

What advantages does the ribosome shunt mechanism oftranslation confer to viral replication? The shunt has clearlyevolved as a mechanism to avoid the problems associated withscanning a highly structured 59 UTR. In this respect, shuntingconfers translational efficiency to the viral mRNA. More spe-cifically, however, ribosomal shunting may preclude or reducethe requirement for the host eIF4F helicase activity to melt thesecondary structures within an mRNA that impede the normalscanning and the AUG site selection process. This would alsorelieve the competition with cellular mRNAs for the limitedamounts of eIF4F present within the host cell. Thus, the ribo-some shunt may facilitate the selective translation of viralmRNA, especially under cellular conditions in which the poolof functional eIF4F becomes limiting. Schneider and col-leagues have investigated this idea by examining the mecha-nisms of selective translation of late adenovirus mRNAs withininfected cells (reviewed in reference 411). Adenovirus inducesthe dephosphorylation of eIF4E and concomitant reduction inthe functional pool of eIF4F during late-stage infection, result-ing in inhibition of host cell protein synthesis (210, 499) (seebelow). However, the translation of late adenovirus mRNAsremain uncompromised, due to the function of the conservedtripartite leader.

The adenovirus late mRNA tripartite leader sequence iscomposed of a linear 59 end followed by regions of stablesecondary structure (91). In uninfected cells, the tripartiteleader can direct the translation of a downstream cistron byboth conventional 59 scanning and ribosome shunting. How-ever, during late-stage adenovirus infection, the translation oflate viral mRNAs is mediated exclusively by the ribosomeshunt (494). Structure-function analysis of the tripartite leaderhas shown that (i) the linear 59 end is essential for the efficientrecruitment of ribosomes when eIF4F is limiting, (ii) the stem-loop structural elements inhibit ribosome scanning during late-stage adenovirus infection, and (iii) the selective translation oflate adenovirus mRNAs is influenced by the actions of one ormore viral gene products. These results, taken together, sug-gest that limitations in eIF4F select for expression of the lateadenovirus gene products through the function of the latemRNA tripartite leader. Stem-loop elements within the tripar-tite leader, along with virus-encoded transacting factors, thendirect the selective translation of late mRNAs through a pro-cess of ribosome shunting, independent of eIF4F helicase ac-tivity. Thus, it appears that the ribosome shunt has evolved thedual functions of allowing the ribosome to avoid the impedi-ments on translation imposed by regions of mRNA secondarystructure and the limitations in the quantity and quality of thehost translation machinery.

Leaky scanning. Translation initiation site selection is de-termined, in part, by the context of the nucleotide sequencesurrounding the first AUG codon encountered by the scanningribosomal subunit. Departure from the canonical accAUGgsequence (the initiation codon is shown in bold letters) oftenresults in the scanning ribosome initiating translation from thisweak AUG at a low frequency or bypassing it completely infavor of a stronger downstream AUG start site (221). ThisAUG selectivity is referred to as leaky scanning. Leaky scan-ning allows the translation of multiple ORFs from a commonmRNA substrate (Fig. 5). The versatility of leaky scanning isquite evident when one considers that each ORF need not bein the same reading frame. Thus, the process of leaky scanningallows the virus to maximize its genome coding capacity andencode functionally distinct proteins from a common mRNA.

Leaky scanning is widely used by viruses and is perhaps bestdefined from studies on retrovirus replication. Human im-munodeficiency virus (HIV) encodes a heterogeneous classof mRNAs that include several multicistronic species. Amongthese are the bicistronic mRNAs encoding the viral Vpu andEnv proteins (368). The ORFs for Vpu and Env are tandemlyarranged such that the Vpu coding region precedes the EnvORF (Fig. 8A). Synthesis of Env is essential for viral replica-tion and takes place through a mechanism of leaky scanningfrom the upstream Vpu ORF (368, 412). Env synthesis re-quires a weak Vpu translation initiation codon. Mutation ofthe weak Vpu start site to a sequence more closely matchingthe canonical start site sequence resulted in suppressed Envtranslation from the bicistronic Vpu-Env mRNA (412). Thus,the weak context of the Vpu initiation codon allows the ribo-some to scan pass the Vpu ORF and to initiate translation atthe downstream Env AUG codon (Fig. 8A). Analyses of HIVmutants in which the vpu gene was deleted or lacked the Vpuinitiation codon revealed a stimulation of Env synthesis (413).Accordingly, vpu mutant viruses exhibited defects in virus re-lease and showed increased syncytium formation in vitro (368).These results support a model for the coordinate expression ofVpu and Env during HIV infection, which is dependent on thepresence of the vpu ORF. By this model, the synthesis of Vpuoccurs inefficiently via a weak initiation codon, perhaps allow-ing Vpu to coordinately accumulate during infection to levelssufficient for its function in late-stage HIV replication. In ac-cordance with the leaky-scanning mechanism, the synthesis ofVpu itself may coordinate Env production by impeding ribo-some scanning to the downstream Env ORF. This model re-mains to be directly examined by analyzing the polyribosomedistribution of the relevant mutant Vpu-Env mRNAs. How-ever, it suggests that the limitations placed on Env translationby the upstream vpu ORF may allow Env expression to coin-cide with late-stage replication events, including virion assem-bly, and release. Thus, while ensuring that both viral proteinswill be produced during HIV infection, translational control byleaky scanning provides for the coordinate expression of Vpuand Env during viral replication.

Frameshifting. As in the example provided by the HIV ge-nome, retroviral genomes exhibit overlapping gene arrange-ments (Fig. 8A). The mouse mammary tumor virus (MMTV)genome exhibits an overlapping gag, pro, and pol gene arrange-ment (72). Translation of these gene products involves theprocess of frameshifting, in which the translating ribosomeshifts position by 11 or 21 nt, resulting in a change of readingframe (reviewed by Gesteland and Atkins [151]) (Fig. 5). Theprocess of frameshifting was first described from studies ofRous sarcoma virus replication (220) and has been extensivelydefined in the MMTV and infectious bronchitis virus (IBV) (acoronavirus)] systems, although it occurs widely throughout



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other eukaryotic RNA viruses (151). Frameshift sites withinthe viral mRNA correspond to heptanucleotide sequences inwhich the mRNA slips 1 base with respect to the tRNAs in theA and P sites on the translating ribosome (40, 108). The frame-shift site, also known as the slippery site, allows the tRNA tomove along the mRNA template by 1 base (forward or back)and reestablish codon-anticodon pairing, resulting in a stable11 or 21 reading frame shift.

Frameshifting is stimulated by the presence of an RNApseudoknot structure located 2 to 4 nt downstream of theslippery site (59, 219, 319). A general model for retroviralframeshifting has been proposed (108), in which the elongatingribosome pauses on the mRNA upon encountering the pseu-doknot structure (Fig. 8B). Ribosomal pausing facilitates therealignment of the slippery-sequence-decoding tRNAs in the21 reading frame. The heptanucleotide sequence that com-prises the slippery site typically conforms to the motif XXXYYYN. Frameshifting occurs at this site through the slippingof two ribosome-bound tRNAs that are translocated from thecurrent reading frame of X-XXY-YYN, to the 21 readingframe of XXX-YYY. In MMTV, translation initiation of thegag-pro mRNA begins at the 59 end of the gag gene (Fig. 8B).The translating ribosomes encounter a slippery site and a pseu-doknot structure near the 39 end of the gag gene. The majorityof the ribosomes read through this region, but approximately25% hesitate at the heptanucleotide site, where the mRNA will

slip backward by 1 nt. This event is stabilized by the newpairing with the two tRNAs in the 21 reading frame. Mean-while, most of the translating ribosomes will terminate to makeGag-Pro but another 10% will slip again at the pro-pol site tomake the requisite Gag-Pro-Pol polyprotein (108).

What are the molecular mechanisms by which the tRNAsand pseudoknot contribute to frameshifting during mRNAtranslation? Evidence has accumulated to indicate that theactual frameshift occurs at the second (underlined) codon ofthe tandem slippery codon pair, XXXYYYN, corresponding tothe ribosome aminoacyl (A) site. Slippery A sites within eu-karyotic viruses correspond to the codon sequence of AAC,AAU, UUA, UUC, and UUU (108). Interestingly, thesecodons are decoded by tRNAs with a highly modified base inthe anticodon loop (185). Thus, it has been suggested thathypomodified variants of these tRNAs may function to pro-mote shifting by being less bulky and therefore more easilymoved within the slippery site (186). However, this idea re-mains controversial, since other researchers have proposedthat frameshifting is mediated by standard cellular tRNAs andis simply dependent on the strength of the codon-anticodontRNA interaction (40, 465). In either case, frameshifting re-quires a pseudoknot structure near the slippery site to stimu-late the frameshifting events.

Recent evidence indicates that the actual secondary struc-ture of the pseudoknot is important for stimulating frame-

FIG. 8. Leaky scanning and translational frameshifting during retroviral mRNA translation. (A) Leaky scanning during HIV mRNA translation accounts forsynthesis of the Vpu and Env proteins. The HIV gene structure (upper) consists of several overlapping cistrons encoded within a heterogeneous array of mRNAs (438).Synthesis of the Vpu and Env proteins (lower diagram) of HIV proceeds via a leaky-scanning mechanism in which the scanning ribosome bypasses the weak vpu AUGcodon (shown) to initiate translation from the env AUG codon (bent arrow). Translation initiation occurs at the vpu AUG codon at a low frequency and may accountfor the stoichiometric ratios of Vpu and Env protein accumulation during HIV infection (413). (B) Translational frameshifting at the gag-pro junction during MMTVmRNA translation. An RNA pseudoknot near the 39 end of the gag gene causes the elongating ribosome to pause at the gag-pro slippery sequence element. As a result,the mRNA slips backward by 1 nt and the ribosome-bound tRNAs mediate new anticodon base pairing in the 21 reading frame (12). Frameshifting is favored by weakcodon-tRNA anticodon base pairing in the original reading frame and strong base pairing in the new reading frame. Synthesis of the entire Gag-Pro-Pol polyproteinis facilitated by a second frameshift at the downstream pro-pol slippery site. Model adapted from reference 12.



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shifting. Analysis of the IBV frameshifting signals clearly dem-onstrated that the pseudoknot causes ribosome pausing.Replacement of the IBV pseudoknot with a simple stem-loopstructure of equivalent base pairs did induce ribosome pausingbut, remarkably, did not stimulate frameshifting (427). Theseresults suggested that ribosome pausing was necessary but notsufficient for frameshifting to occur and support the hypothe-ses that (i) conservation of pseudoknot structure is essentialfor frameshifting and (ii) pseudoknot interactions with specifictrans-acting factors may promote the frameshift events. Atomicmodeling of the MMTV gag-pro pseudoknot supports the form-er hypothesis, in that this pseudoknot does not have coaxiallystacked helices but, rather, assumes a wedge conformation in-duced by an A nucleotide between the helices (418). Structure-function analyses of the MMTV pseudoknot revealed that thisA nucleotide was essential for stimulating frameshifting activ-ity. Structural analyses of other viral pseudoknots should pro-vide further insight into the contribution of pseudoknot se-quence and structure in ribosome frameshifting.

Control of termination and reinitiation. Translational con-trol by reinitiation involves two or more tandemly arrangedORFs on a common mRNA. In the simplest model of reini-tiation, a short uORF controls the translation of the majordownstream ORF by impeding ribosome scanning (reviewedby Geballe [146]). In this sense, the uORF commonly asserts asuppressive effect upon translation of the downstream ORF.However, and as in the CaMV 35S RNA translation, excep-tions to this rule do apply. As described above, the sORFAuORF of CaMV actually plays a stimulatory role in translationof downstream ORFs within the 35S RNA (195). An exampleof viral use of reinitiation to negatively control translation froma downstream cistron comes from studies of cytomegalovirusreplication. During cytomegalovirus infection, expression ofthe gp48 product of the polycistronic viral gpUL4 mRNA iscoordinately controlled to reach peak levels during late-stageviral replication. gp48 is translated from the third of threecistrons within the gpUL4 mRNA (146, 313). Gelballe and col-

leagues have determined that coordinate control of gp48 ex-pression is mediated through the actions of the second gpUL4uORF (uORF2) (147). Remarkably, the uORF2 inhibitoryeffect on gp48 translation is dependent upon the sequence ofuORF2 (48, 85); introduction of uORF2 missense mutationsseverely diminished the inhibitory signal upon gp48 transla-tion, while introduction of mutations that preserved the codingcontent of uORF2 led to retention of gp48 translational inhi-bition. In vitro and in vivo expression studies revealed that thetranslational control actions of uORF2 (i) function exclusivelyin cis to repress gp48 synthesis through ribosome stalling at theuORF2 termination codon and (ii) are mediated through in-terference of uORF2 translation termination by the uORF2peptide product itself. Analysis of uORF2 translation revealedthat the 20-kDa peptide product remained bound to the ribo-some complex as a peptidyl-tRNA covalently linked to tRNApro,which decodes the uORF2 carboxyl-terminal codon (49). Re-cent studies have now demonstrated that the uORF2 peptidyl-tRNApro blocks its own hydrolysis and ribosome release toremain stably bound to the ribosomal complex (50). Theseresults suggest that inhibition of uORF2 peptidyl-tRNApro hy-drolysis blocks the translation of gp48 by creating a barrier thatobstructs ribosome scanning to the downstream gp48 ORF(Fig. 9).

If uORF2 blocks gp48 translation, what facilitates synthesisof the gp48 protein during CMV infection? Sequence analyseshas shown that the initiator AUG codon of uORF2 is pre-sented within a “weak” context for optimal translation initia-tion (147). Accordingly, it was found that the uORF2 AUGcodon is recognized by the ribosomal complex only at a lowfrequency and is actually bypassed by a leaky-scanning mech-anism in favor of initiating translation from the gp48 startcodon. Alteration of the uORF2 initiation codon to within anoptimal context for translation initiation results in nearly acomplete block in gp48 synthesis (48). Together, these resultssupport a bipartite model for the control of gp48 translation byuORF2 (Fig. 9). It is not clear how uORF2 peptidyl-tRNApro

FIG. 9. CMV control of gp48 synthesis by inefficient termination of uORF2. During CMV infection the viral gp48 glycoprotein is translated from the third of threecistrons (rectangles) within the gpUL4 mRNA. 1, gp48 synthesis is facilitated by a leaky-scanning mechanism in which the scanning ribosome bypasses the weak upstream AUGcodons within the gpUL4 mRNA to initiate translation at the gp48 AUG (indicated by arrow); 2, translation initiation at the uORF2 AUG occurs at a low frequency and resultsin control of gp48 translation. Synthesis of the uORF2 peptide produces a stable peptide-tRNApro-ribosome complex that prevents peptide release and stalls theelongating ribosome at the uORF2 termination codon. As a result, ribosome scanning and reinitiation at the downstream gp48 AUG codon is blocked.



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blocks its own hydrolysis and release from the uORF2 termi-nation site. Possible explanations may be found by examiningthe influence of the uORF2 product on the function of trans-lation elongation and release factors.

Another example of reinitiation control comes from studiesof reovirus translation. Reoviruses are double-stranded RNA(dsRNA) viruses with a segmented genome. The S1 mRNA ofreoviruses is bicistronic and encodes the s1 capsid protein andthe s1 nonstructural protein (409, 417). Initial analyses of S1mRNA translation revealed that it was translated inefficientlycompared to other reovirus mRNAs (345). Subsequent in vitrostudies showed that ribosomes paused at several positions onthe S1 mRNA relative to the S4 mRNA, suggesting that trans-lating ribosomes were less evenly distributed along the codingregion of the inefficiently translated S1 mRNA than of theefficiently translated S4 mRNA (97). These results were sup-ported by in vivo studies in which the distribution of translatingribosomes on polyribosome-bound reovirus S1 and S4 mRNAswas examined in reovirus-infected cells (98). The pattern ofribosome pausing in vivo showed that ribosomes were lessevenly distributed along the poorly translated bicistronic S1mRNA. Consistent with a model of S1 mRNA translationalcontrol by reinitiation, expression of the downstream S1 ORFwas significantly increased by mutation of the upstream AUGcodon to a less favorable context for translation initiation(106). Interestingly, however, the synthesis of the upstream S1mRNA translation product was not decreased by the samemutations. Identification of differential codon usage betweenthe S1 ORFs suggests that the translation efficiency of the S1uORF may be due to codon usage that confers a low elonga-tion rate through the utilization of low-abundance tRNAs(106). The diminished elongation rate of the S1 uORF maythen limit the efficiency of reinitiation and synthesis of thedownstream S1 cistron. Studies aimed at understanding theinfluence of differential codon usage on the elongation ratemay uncover additional examples of this type of translationcontrol among eukaryotic viruses.

As described in the examples cited above, control of re-initiation has been attributed largely to processes inherentwithin the 59 UTR of the viral mRNA. Analyses of alfalfamosaic virus (AMV) replication now suggests that the 39 UTRmay likewise play an important role in translation reinitia-tion and the efficiency of viral protein synthesis. The single-stranded RNA genome of AMV is capped but not polyadenyl-ated. Translation studies have revealed that AMV RNAs areefficiently translated in spite of lacking the traditional poly(A)tail and the advantages to RNA stability and translation af-forded to polyadenylated transcripts (157, 200). In contrast tothe many viruses that induce host translational shutoff duringinfection, AMV infection, AMV infection is not associatedwith a decrease in host protein synthesis (149). How, then, doAMV mRNAs adequately compete for available translationfactors? Early evidence suggested that the 39 UTR of the AMVcoat protein played a stimulatory role in mediating coat pro-tein synthesis (399, 496). Examination of coat protein mRNAtranslation in vitro and in vivo revealed this mRNA to beefficiently translated even in the presence of large quantities ofa cellular mRNA competitor. A functional role for the 39 UTRin coat protein mRNA translation was demonstrated by con-ducting similar experiments with mutant mRNAs lacking the 39UTR; loss of the 39 UTR consistently reduced the efficiency ofcoat protein synthesis without altering mRNA stability (177,399). Interestingly, it was found that the 39 UTR was requiredfor assembly of the coat protein mRNA into polyribosomecomplexes, indicating that the 39 UTR was an important de-terminant for ribosome binding (177). Mutagenesis studies

were used to identity the 39 UTR nucleotide sequence elementGAUG as an important determinant in AMV coat proteinsynthesis. This tetranucleotide sequence encompasses an initi-ation codon downstream from the coat protein terminationcodon and is thought to stimulate coat protein synthesisthrough a process of reinitiation (177). With this model, reini-tiation would facilitate ribosome-mRNA interaction and con-tinued coat protein synthesis.

How might reinitiation within the 39 UTR actually contrib-ute to increased translational efficiency? One possibility is thatreinitiation may retain the mRNA within the pool of activeribosomes, thereby increasing the probability of 59 UTR-ribo-some interactions and promoting further rounds of authentictranslation initiation. On the other hand, the viral 39 UTR maystimulate coat protein translation through a process indepen-dent of reinitiation, although this idea remains inconsistentwith experimental evidence. In this case, it remains possiblethat specific 39 UTR-protein interactions may impart increasedtranslational efficiency.

Functional recoding. In addition to frameshifting, virusespartake in functional recoding whereby translation proceedsthrough an in-frame termination codon (Fig. 5). This occursthough a process of redefining the termination codon to en-code glutamine at UAG or tryptophan or selenocysteine atUGA (151). Functional recoding has been extensively studiedin the Moloney murine leukemia virus (MuLV) system. MuLVredefines the stop codon at its gag-pol junction through theinsertion of glutamine at the UAG stop codon. This allows thetranslating ribosomal complex to read through the gag-poljunction and synthesize the Gag-Pol polyprotein. During viralreplication, the ribosome reads through the MuLV gag-polstop codon 5% of the time, and this exact frequency seems tobe essential for replication (491). If the UAG codon is re-placed with an in-frame GAG codon, no viral particles areformed (111). In contrast, replacement of the native gag-polstop codon with either the UAA or UGA stop codon permittedtranslational readthrough with similar efficiency. These resultssuggest that MuLV utilizes functional recoding as a mecha-nism to control the level of Gag-Pol polyprotein synthesisduring the course of infection.

Moreover, it appears that redefining the stop codon is notcodon specific, suggesting that other elements within the viralmRNA are responsible for translational readthrough. It is nowclear that sequences downstream of the stop codon are neces-sary for functional recoding. In MuLV, this includes a pseu-doknot sequence that appears to stimulate the recoding pro-cess (112, 482) (Fig. 10). Structure-function analyses of theMuLV pseudoknot revealed several interesting features re-quired for stimulating recoding, including (i) specific nucleo-tide sequence within the spacer region between the stop codonand the pseudoknot; (ii) nucleotide conservation within stem-loop 2 of the pseudoknot; and (iii) a nonhelical pseudoknotstructure (482). These results support the idea that recodingmay be dependent upon recruitment of trans-acting factors tothe termination site, possibly mediated through sequenceswithin the spacer region between the stop codon and the pseu-doknot and/or the pseudoknot structure itself. Identification ofsuch factors may have implications for the development offuture antiviral drugs, since disruption of the recoding processresults in a block in virion formation within the infected cell(111).

Coupling the Virus Life Cycle to Translational Control

Translation control programs in eukaryotic cells play impor-tant roles in governing cellular metabolism. In many cases,



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these programs are implemented in response to specific envi-ronmental cues. Viruses, specifically the more complex DNAviruses, including the poxviruses, papillomaviruses, and her-pesviruses, have similarly incorporated into their own life cy-cles translational control programs that play important roles inreplication, latency, and virulence. In particular, recent evi-dence from studies of HSV replication indicates that transla-tional control programs are implemented by the virus to (i)facilitate host shutoff, (ii) control global and specific viral geneexpression, (iii) maintain or exit latency, and (iv) overcome thehost antiviral response. In this section, we use the herpesviruslife cycle to illustrate how viruses utilize translational controlprograms to direct replicative decisions and how this transla-tional programming contributes to the control of viral replica-tion.

The herpesviruses. The human herpesviruses establish la-tent infection in either neural cells (HSV and varicella-zostervirus) or hematopoietic cells (Epstein-Barr virus and cytomeg-alovirus) with negligible damage to their respective host. La-tency permits viral persistence in the face of an active immuneresponse. In response to certain stimuli, the viruses may peri-odically reactivate from latency throughout the life of the hostto enter the productive phase viral life cycle, which shedssufficient virus progeny to infect new hosts. This life-style isbound to impose a distinctive set of evolutionary pressures onthe control mechanisms regulating herpesviruses gene expres-sion. Thus, herpesviruses present a challenging and attractivemodel system for studying the ever-complex mechanisms ofviral gene expression and regulation at the level of translation.Furthermore, mutant herpesviruses deficient in a particularfunction can be isolated or genetically engineered with dele-tions in certain genes to assess the roles of specific virus-encoded proteins in viral translation and replication in thehost. In the following sections, we examine how the alphaher-pesviruses HSV-1 and HSV-2 have implemented control ofviral and host translation into their latent and productivephases and discuss how translational control of HSV mRNAmight contribute to viral pathogenesis.

Because HSV gene expression is coordinately modulatedduring a productive infection, the viral genes can be catego-rized into three kinetic classes: immediate-early (a), early (b),

and late (g) genes. a genes are transcribed in the absence of denovo protein synthesis; this process peaks at 2 to 4 h postin-fection, and the transcripts continue to accumulate until late ininfection. Most products of a genes are potent transcriptionaltrans-activators that cooperate to activate the transcriptionof b and g genes. b genes encode proteins required for HSVDNA synthesis, as well as a number of auxiliary replicationfactors. Viral structural proteins are the products of g genes,whose expression occurs at the onset of DNA synthesis. Whilemuch research on HSV has centered on the transcriptionalevents responsible for the differential expression of a, b, and ggenes, accumulating evidence indicate that translational mech-anisms are also important for HSV gene expression.

HSV and the shutoff of host cell protein synthesis. Likeother cytolytic viruses, HSVs are thought to facilitate theirreplication by preferentially producing viral proteins at theexpense of host cell gene expression. In tissue culture cellsinfected with HSV-1 or HSV-2, host protein synthesis andmRNA levels decrease by approximately 90% within 3 hpostinfection and viral proteins dominate thereafter (424).This remarkable feature of the shutoff of host protein synthesisinduced by HSV infection, presumably to alleviate competitionfor precursors, is a multistep process that involves severalmechanisms and can be separated into two stages: primaryshutoff and secondary shutoff. Primary shutoff, which is char-acterized by rapid disintegration of preexisting polyribosomesand degradation of preexisting cellular and viral mRNAs, oc-curs very early after HSV infection in the absence of de novoprotein synthesis. In contrast, secondary shutoff takes placelater in the course of infection and requires viral gene expres-sion.

Despite extensive research efforts, the exact mechanismsresponsible for the shutoff events during HSV-1 infection arepoorly understood, but encouraging progress has been madeover the past few years. In the primary shutoff, at least one viralfactor, the virion host shutoff (VHS) protein, is necessary formRNA destabilization, and this may, at least in part, accountfor the disassociation of polyribosomes (280). Encoded byHSV gene UL41, the VHS protein is a 58-kDa phosphoproteinlocated between the capsid and envelope regions of the virion(called tegument) and is delivered into the cytoplasm of newlyinfected cells. How does VHS function to specifically inducemRNA degradation? Apart from having limited homology to asmall segment of PABP (389), the primary sequence of theVHS protein provides little clue to its function. Despite thelack of any primary sequence similarity to known RNases,several lines of evidence suggest that VHS is associated withRNase activity: (i) incubation of polyribosomes from unin-fected cells with postpolysomal (S130) supernatant from HSV-infected cells, but not S130 from uninfected cells or from cellsinfected with a VHS-defective virus, resulted in rapid degra-dation of stable mRNAs; (ii) crude extracts from host shutoff-competent virions or reticulocyte lysate containing wild-typeVHS protein displayed enhanced RNase activity, while VHSmutants did not; and (iii) antibodies against VHS protein in-hibited the RNase activity of wild-type VHS protein in thecell-free reactions. However, there is as yet no direct evidence,obtained using highly purified proteins, to demonstrate thatwild-type, but not mutant, VHS protein itself indeed containsRNase activity in vitro. Thus, the possibility that the VHS pro-tein works in conjunction with another factor(s) with RNaseactivity has not been entirely excluded.

The mechanism(s) by which the VHS protein specificallytargets mRNA for degradation is not known. PolyadenylatedRNAs are degraded faster than nonpolyadenylated substrates(225), and deadenylated mRNAs congregate in HSV-infected

FIG. 10. Functional recoding at the MuLV gag-pol junction. Synthesis of theMuLV Gag protein terminates at the gag stop codon (top). However, approxi-mately 5% of the time, the elongating ribosome will read through the gag stopcodon to produce the Gag-Pol polyprotein (bottom). During this process, the gagstop codon is redefined to encode glutamine and is shown by the presence oftRNAGln at the redefined UAG codon. Stop codon redefinition is dependent onspecific downstream sequences and a 39-proximal pseudoknot structure. Theelongating ribosome eventually melts out the pseudoknot to complete Gag-Polsynthesis. Low-frequency gag-pol stop codon redefinition is essential for MuLVreplication. Figure adapted with modification from references 12 and 108.



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cells (180). Thus, it is conceivable that the VHS protein rec-ognizes polyadenylated mRNAs, perhaps through the putativepoly(A)-binding region of VHS. In this context, it would beinteresting to test the effect of mutating the conserved residuesin this region on HSV-induced mRNA degradation and trans-lational arrest. Furthermore, the ability of the VHS protein tobind poly(A) mRNA in vivo (for example, through UV cross-linking procedures) or the RNA sequences or structural re-gions recognized by VHS have not been determined. Alterna-tively, the VHS protein may interact with the PABP complexfor localized poly(A) cleavage of mRNA. This possibility isconsistent with the observation that mRNAs in infected cells orin cell-free reaction mixtures are preferentially degraded overprotein-free RNAs, suggesting that the VHS protein mightachieve specificity through interaction with a factor(s) presentin the ribonucleoprotein (RNP) complex. Therefore, the iden-tification of VHS-interacting factors may also provide insightsinto the mechanistic action of the VHS protein. At any rate,considering the specificity of the VHS protein and the gener-ation of discrete decay mRNA intermediates, VHS probablyoperates, either on its own or in cooperation with anotherfactor(s), by cleaving mRNA molecules at one or a few criticalsites, such as poly(A), that normally function to protect mRNAfrom destruction by host RNases.

The ICP27 protein of HSV-1 is thought to play an importantrole in the secondary shutoff of host protein synthesis (180).A nuclear phosphoprotein of 63 kDa, ICP27 was originallyknown for its capability to both stimulate and repress theexpression of different target genes. Subsequent studies dem-onstrated that ICP27 is also capable of interfering with hostcell splicing, causing a reduction in the levels of several cellularspliced transcripts and an accumulation of pre-mRNA in thenucleus during infection (180, 181). Furthermore, ICP27 ex-pression causes a dramatic redistribution of the splicing smallnuclear RNPs and other splicing factors during HSV-1 infec-tion (406). Finally, an interaction between ICP27 and the splic-ing machinery has been demonstrated (181, 406). On the basisof these studies, it has been hypothesized that ICP27-mediatedimpairment of host cell splicing may contribute to the second-ary shutoff, because unspliced cellular transcripts remain in thenucleus, where they become degraded. This leads to fewercellular transcripts being exported to the cytoplasm for trans-lation, and thus selective synthesis of virus-specified proteins isfavored, since most viral transcripts are not spliced.

Selective repression of mRNA translation initiation duringHSV infection. HSV induces shutoff of most host protein syn-thesis. However, a few remaining cellular mRNAs, whose pro-tein products presumably play an important role in the survivalof the virus within the host, are continuously being translatedafter infection (285). This paradox raises some important ques-tions. (i) What is the function and nature of these cellularmRNAs? (ii) Is the sustained translation of cellular mRNAsinduced by HSV or an inherent feature of the mRNAs? (iii)What is the mechanism of the persistence of translation ofthese mRNAs after infection?

Recent studies demonstrate that at least one set of cellularmRNAs that are persistently translated after HSV-1 infectionencode ribosomal proteins (164, 425). An analysis of ribosomalprotein mRNA expression across a polyribosome gradient re-vealed that there is a discernible shift between the untranslatedsubpolysomal (prepolysomal) fraction to the polyribosomalfraction as infection proceeded (164). It is known that the 59leader sequence of vertebrate ribosomal protein mRNAs con-tains a terminal oligopyrimidine tract (known as the 59 TOPelement) that is sufficient to confer translational regulation andmigration between the polyribosomal fractions (13). The spe-

cific recruitment of 59 TOP mRNAs by ribosomes is closelyassociated with an increase in phosphorylation of ribosomalprotein S6 (231, 232, 456). In this regard, S6 proteins in pre-existing ribosomes were phosphorylated to greater extent thanwere those found in newly assembled ribosomes in infectedcells (164). This suggests that translation of ribosomal proteinmRNAs may occur preferentially on preexisting ribosomes.Interestingly, in parallel with this study, a progressive shiftof b-actin and GAPDH mRNAs from polyribosomes to 40Ssubunits was observed during the course of infection. Thisphenomenon appeared to be independent of VHS-mediatedmRNA degradation, since the protein did not affect mRNArecruitment by polyribosomes. With the caveat that only twocellular mRNAs have been examined, these studies suggestthat HSV-1 may employ an additional strategy to selectivelysuppresses host mRNA translation, namely at the initiationstep.

Other mechanisms may also contribute to the persistence ofribosomal protein synthesis. In addition to S6 ribosomal pro-tein, HSV-1 infection induces phosphorylation of at least twoother proteins, including the product of the US11 late gene(90). Furthermore, the possibility that an increase in the elon-gation rate of translation might also account for the sustainedtranslation of ribosomal protein mRNAs has not been elimi-nated. In this regard, two HSV-1 proteins, the trans-activatorICP0 protein (10) and the protein kinase encoded by theU(L)13 gene (257), interact with and phosphorylate elongationfactor-1 delta (EF1-d), respectively. However, a role for EF1-dphosphorylation in HSV-1 replication has not been demon-strated.

Selective translation of HSV mRNA. How does HSV achieveselective viral translation during the shutoff of protein synthe-sis? In VHS-induced shutoff, in which both cellular and viralmRNAs undergo concomitant degradation, HSV would needto ensure that the viral mRNAs continue to accumulate aftercellular mRNAs have been degraded. The potency and indis-criminate nature of the VHS activity suggests that it wouldhave to be negatively controlled in a temporal fashion duringthe course of infection. Evidence for this hypothesis came froma study demonstrating that the virion trans-activator VP16,which forms a specific complex with VHS in the infected cell,is capable of suppressing VHS activity (282). Specifically, viralprotein synthesis and mRNA levels were significantly reducedat intermediate times after infection with a VP16 null mutantvirus. Additionally, a stably transfected cell line expressingVP16 was refractory to VHS-induced host shutoff of proteinsynthesis. Although it remains to be shown, the VHS bindingfunction of VP16 is likely to be important for inactivating VHS,either by masking one or more functional domains, inducing aconformational change, or by targeting VHS into the nucleusand/or the virion assembly pathway. Moreover, the mechanismby which the VP16-VHS interaction is modulated is notknown. Finally, it is noteworthy that two other viral factors areinvolved in HSV-induced host shutoff: the virion-associatedprotein kinase encoded by the U(L)13 gene (358) and theICP22 protein encoded by the US1.5 gene (349). The possibil-ity that these gene products may mediate host shutoff by reg-ulating VHS and/or VP16 function will undoubtedly be ex-plored in forthcoming studies.

Another strategy by which HSV may exert selective transla-tion of viral mRNAs over host mRNAs is suggested by recentstudies that demonstrate a role of the ICP27 protein in theexport of HSV-1 intronless mRNAs (405). Thus, ICP27 ap-pears to mediate preferential viral translation via two mecha-nisms: (i) it impedes the translation of cellular mRNA bypreventing the export of this mRNA to the cytoplasm through



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the impairment of host splicing, and (ii) it binds viral tran-scripts and delivers these RNAs to the cytoplasm for transla-tion. However, several key aspects will have to be elucidated infuture studies to strengthen the proposed dual role of ICP27.These include assessing (i) the functional significance (an ef-fect on splicing) of the interactions and changes of ICP27 andsmall nuclear RNPs, (ii) the specificity of ICP27 RNA binding,and (iii) the interaction of ICP27 with the cellular nuclearexport complex.

Finally, the observations that ribosomal proteins are persis-tently synthesized and new ribosomes are assembled afterHSV-1 infection might suggest another mechanism for thepreferential translation of viral mRNAs. Because new ribo-somes contain underphosphorylated S6 ribosomal protein,they are presumably less effective in the translation initiationof mRNAs that possess 59 TOP sequences (reviewed in refer-ence 232). Thus, viral mRNAs which lack 59 TOP sequencesmay be selectively initiated by newly synthesized ribosomesover cellular mRNAs containing 59 TOP. Although the idea isnot unreasonable, especially since reinitiation of translationprogressively becomes a limiting factor during shutoff (10,285), it remains to be supported by any experimental evidence.

Inhibition of eIF2a phosphorylation during HSV infection.Cells modulate the synthesis of proteins in response to externalstimuli, including viral infection, through the modification oftranslation factors. Phosphorylation of eIF2a is perhaps thebest-characterized mechanism by which this occurs, particu-larly within the context of virus infection. As described in detailin “Viral modification of translation factors” (below), viralreplication produces highly structured viral transcripts in theform of dsRNA that can bind to and activate the host PKR,which in turn phosphorylates eIF2a (131). As a result, thecellular translational machinery is incapacitated and viral pro-tein synthesis and replication are restricted within the infectedcell. Accumulating evidence now indicate that HSV-1, likemany viruses (see Table 4), has evolved ways to circumvent thevirally induced translational block by counteracting PKR func-tion (Fig. 11).

Initial evidence that HSV-1 can antagonize PKR-mediatedtranslational arrest stemmed from studies of mutant virusesthat lack the g134.5 gene. These viruses fail to grow on manyhuman malignant neuronal cells, which displayed increasedPKR and eIF2a phosphorylation, as well as premature shutoffof protein synthesis late in infection. The phenomenon ap-peared to be independent of VHS function and mRNA deg-radation (62, 379). Interestingly, an unknown 90-kDa phospho-protein (p90) coprecipitated with anti-PKR antibody fromlysates of cells infected with g134.52 viruses. While the func-tion of p90 is not known, the correlation between p90 phos-phorylation and the premature shutoff of protein synthesissuggests that it may play a positive role in modulating PKRactivity in phosphorylation of eIF2a. It was thought that theg134.5 gene product might inhibit protein kinase activity byblocking the interaction between p90 and PKR. However, sub-sequent studies demonstrated that the g134.5 protein operatesthrough a different mode of action. Using the yeast two-hybridapproach, He et al. (190) found that the g134.5 protein asso-ciated with the cellular protein phosphatase 1a (PP1a). Fur-ther, the g134.5 protein formed a complex with PP1a in HSV-1-infected cells, and fractions containing the complex werecapable of dephosphorylating purified eIF2a. Thus, the g134.5protein is likely to function as a regulatory or targeting subunitof PP1a to redirect the phosphatase to dephosphorylate eIF2a,therefore neutralizing PKR activity. However, it is not clearhow the g134.5 protein guides PP1a to eIF2a; an interactionbetween g134.5 and eIF2a has not been demonstrated. Inter-estingly, we have recently obtained evidence that PP1a canalso directly inhibit PKR function by binding to and dephos-phorylating PKR (S.-L. Tan and M. G. Katze, unpublisheddata). Whether dephosphorylation of PKR by PP1a is alsotriggered by HSV-1 infection or other signals is not known.Furthermore, it will be important to identify the regulatorysubunit of PP1a that targets the phosphatase to PKR (Fig. 11).

The story becomes more complicated with recent studiesdescribing the isolation of second-site suppressor mutant vi-ruses that lack the g134.5 gene (53, 338). These variant viruses

FIG. 11. Translational control during HSV-1 infection. 1, Infection with HSV-1 induces a rapid shutoff of host cell protein synthesis, due in part to the actions ofthe viral VHS and ICP27 proteins, which indirectly affect mRNA translation. VHS-induced translational shutoff may be directly modulated through the viral VP16protein. 2, Repression of VHS by VP16 may contribute to the selective translation of viral mRNAs. The viral U(L)13 and ICP0 proteins may similarly modulate viralmRNA translation by phosphorylating and binding, respectively, to host EF1-d. During infection, HSV-1 ensures that the host cell remains translationally competentby blocking the phosphorylation of eIF2a. Disruption of eIF2a phosphorylation may occur through the actions of the viral Us11 protein, which prevents PKR activation,and by the viral g134.5 gene product, which directs the dephosphorylation of eIF2a by PPIa. Finally, viral modulation of ribosomal protein S6 phosphorylation maycontribute to the sustained translation of host 59 TOP mRNAs, including those that encode ribosomal proteins. Sustained ribosomal protein synthesis and disruptionof eIF2a phosphorylation facilitates HSV persistence by supporting viral mRNA translation.



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regained the ability to grow on otherwise nonpermissive neu-ronal cells and contained additional mutations that affect adistinct viral genetic element, the SUP locus. Moreover, dele-tion of the SUP locus prevented the accumulation of phosphor-ylated eIF2a. Consequently, extragenic suppressor g134.5 mu-tants could sustain protein synthesis and multiply on cells thatfailed to support replication of the parental the g134.5 variants.As it turns out, these dominant suppressor alleles compensatedfor the loss of the g134.5 function by overproducing a viralRNA-binding, ribosome-associated protein (US11) that reducedPKR activation (343). Taken together, these results suggestthat HSV-1 encodes at least two strategies (US11 and g134.5)to modulate cellular translation by targeting both PKR andeIF2a (Fig. 11). Interestingly, the g134.5 protein contains aregion of significant homology to the cellular proteinGADD34, which is induced in response to agents that promotegrowth arrest, DNA damage, and differentiation. Indeed,GADD34 could also interact with PP1a and could functionallyreplace g134.5 in prolonging late-protein synthesis in infectedcells (189). These studies suggest that signals that trigger dif-ferentiation, growth arrest, and DNA damage may be inti-mately linked to translational control.

Implications of viral modulation of translation in HSVpathogenesis and disease treatment. An important questionthat has remained unanswered in the study of HSV pathogen-esis concerns how the viruses undergo latency in their host. Itis unanswered because of the lack of a reliable cell culturesystem to support latent HSV infection. What is known from insitu hybridization studies of latently infected neuronal cells isthat the latent state is characterized by the transcription ofspecific colinear transcripts. These RNAs, known as latency-associated transcripts (LATs), persist despite the absence ofvirtually all gene expression. Although experiments with LAT-negative mutants showed that the LATs are not required forHSV-1 lytic replication and establishment or maintenance oflatency, they appear to be necessary for efficient in vivo reac-tivation in infected animal models. Thus, there is great interestin determining the stage of latency at which the LATs modu-late, their mechanisms of action, and the exact sequences re-sponsible for reactivation. In this regard, Steiner and col-leagues have recently reported that the LATs are associatedwith polyribosomes in vitro and during latent in vivo infection(158, 159). These observations are highly suggestive of trans-lation of a functional LAT protein, although efforts to demon-strate the presence of HSV-1 LAT protein products in latentlyinfected tissues have been futile to date. To begin to gain anunderstanding of how translational control may play a role inHSV reactivation and thus contribute to viral pathogenesis, itwill be critical to identify mechanism(s) of ribosome binding tothe LAT transcripts and to characterize the LAT proteins.

Finally, the cytolytic and neurotropic properties of HSV-1render this virus a potential tumoricidal agent for destroyingmalignant cells in the central nervous system. In this regard,HSV-1 g134.5 mutant viruses, which are attenuated and non-neurovirulent in animals, are able to discriminate betweennormal and malignant cells (8). However, these variant virusesgrow poorly on neuronal tumors, which imposes a major lim-itation on their effectiveness in destroying tumor tissue. Thesuppressor mutants of the g134.5 allele, which have regainedthe ability to grow on neoplastic cells but retained the atten-uated phenotype of the g134.5 parent virus, may represent aprototype virus to destroy malignant cells in the CNS. Further-more, the use of attenuated HSV as a vector for gene therapyin the study and treatment of neurodegenerative diseases isunder investigation.

Recruitment of Host Factors for the EfficientTranslation of Viral mRNA

The previous examples of HSV-host interactions and theresulting modification of host factors reflects a common themeamong viruses, which target and modify host process to facil-itate viral replication. Indeed, the pressures of translationaldependence on viral replication have resulted in a wide varietyof viral strategies to maximize translational efficiency. Suchstrategies often involve recruitment of specific host factors thatfunction to improve the efficiency or mediate the selectivity ofviral mRNA translation. This section discusses how virusesrecruit and utilize specific host factors, in addition to the con-served repertoire of canonical translation factors, to facilitateefficient mRNA translation during infection. As presented be-low, recruitment of host factors to the viral mRNA is broadlyused in both cap-dependent and cap-independent translationalstrategies. This section will begin by examining the host factorsthat are recruited to the IRES element and the viral 39 UTRand discussing the roles that such proteins may play in medi-ating viral mRNA translation and mitigating virus host range.This is followed by a detailed examination of how influenzavirus ensures the efficient and selective translation of itsmRNAs though a process of host factor recruitment and mod-ification.

IRES binding proteins and proteins that bind the viral 3*UTR. As the functional element for cap-independent transla-tion initiation, the IRES promotes viral mRNA translationthrough the recruitment of canonical translation factors to theinitiation site (223). In addition, it is now known that severalnoncanonical factors are recruited to the IRES that stimulate,or in some cases repress, IRES-mediated translation. Struc-ture-function analyses have provided insights into the roles ofIRES-binding proteins in viral replication and have identifiedsequences within the IRES that direct interaction specificity(162, 238, 239, 394) (reviewed by Belsham and Sonenberg [23],Jackson and Kaminski (223), Hellen and Wimmer [193], andEhrenfeld [101]). Meanwhile, protein interactions with the vi-ral 39 UTR have similarly been implicated in providing trans-lational efficiency and selectivity during viral infection (122,209, 218, 302, 360, 496). A role for 39 UTR-binding proteins inviral mRNA translation may reflect the efforts of viruses totake full advantage of the translational efficiency provided bythe closed-loop translation model.

The early observations from in vitro studies revealed thatefficient translation from enterovirus or rhinovirus IRES inrabbit reticulocyte lysate (RRL) required supplements derivedfrom HeLa cell extracts (41, 99). In contrast, the cardiovirus-aphthovirus IRES conferred efficient translation in nativeRRL. These results were significant in that they indicated thatIRES translation required a specific trans-acting factor(s) sup-plied by the host cell, pointing the way to the identification ofIRES-binding proteins. Moreover, these observations impli-cated IRES-binding proteins in the determination of virus hostrange specificity. IRES-binding proteins have since been iden-tified through a combination of gel mobility shift assays and theuse of UV light to cross-link RNA probes with resident cyto-plasmic factors within cell extracts. Several distinct cellularIRES-binding proteins have been identified and characterized(reviewed in reference 23). Among the best-characterizedIRES-binding proteins are the systemic lupus erythematosusautoantigen (La) and PTB (36, 191, 194, 328, 330). Both pro-teins specifically bind the poliovirus IRES.

La is a 52-kDa RNA-binding protein that is known to inter-act with RNA polymerase III-transcripts and to play a role inthe transcription termination reaction (160). The La protein



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resides predominantly within the nucleus of uninfected cellsbut, importantly, is redistributed to the cytoplasm during pi-cornavirus infection (330). Structure-function analyses haveidentified nucleotides 559 to 624 of the poliovirus RNA as themajor binding site for the La protein (328). This region mapsto within the polypyrimidine tract of the poliovirus IRES (Fig.6). The best evidence in support of a functional role for the Laprotein in poliovirus translation comes from experiments inwhich RRL was supplemented with purified recombinant Laprotein. Addition of recombinant La stimulated poliovirustranslation to an efficiency similar to that observed when RRLwas supplemented with HeLa extract (330). However, the con-centration of recombinant La greatly exceeded the level of Lawithin HeLa extracts, suggesting that (i) recombinant La wasonly partially active or (ii) additional factors may participatewith La to promote poliovirus translation. It should be notedthat the La protein also binds to the 59 UTR of influenza virusand HIV RNA, where it may also play roles in facilitatingefficient viral protein synthesis (361, 443). Thus, the functionalrole of La in viral protein synthesis may not be limited topromoting IRES-mediated translation but may reflect a moregeneral function. Although a direct biochemical role for La inIRES-mediated translation remains to be demonstrated, it ispostulated that La may function as an RNA chaperone tomaintain RNA structure in a conformation that favors trans-lation.

PTB is a 57-kDa cellular protein that plays a role in RNAsplicing (304). PTB binds to multiple sites within the poliovirusIRES (191), but a direct role for PTB function in poliovirustranslation has yet to be demonstrated. PTB also binds withhigh affinity to the EMCV IRES (229) and, more recently, tothe IRES of HCV (3, 4, 217). Major PTB-binding sites havebeen identified within stem-loop H and the polypyrimidinetract of EMCV (229) (Fig. 6). Similar to the La protein, PTBis postulated to function as an RNA chaperone, where it maystabilize IRES structure in a translation-competent conforma-tion. Evidence to support this idea comes from analyses ofEMCV RNA that possessed point mutations within the stem-loop H PTB-binding site (229). Mutations that disrupted stem-loop base pairing prevented PTB binding and abrogated IRESfunction. Meanwhile, compensatory mutations that restoredthe native stem-loop conformation restored both PTB-bindingand IRES function. A direct role for PTB in EMCV IRES-mediated translation was suggested from competition experi-ments in which PTB was functionally depleted from in vitrotranslation reaction mixtures by the addition of competitorRNA that contained a PTB-binding site (38). Addition of com-petitor RNA was found to specifically inhibit EMCV transla-tion, and the addition of exogenous PTB relieved this compe-tition to restore EMCV translation. However, recent resultsfrom Kaminski and Jackson (239) suggest that PTB bindingmay not be an absolute requirement for EMCV translationbut, rather, is conditional upon the structure of the major PTBbinding site within the EMCV IRES.

In addition to La and PTB, several other factors have beenidentified that may play a role in IRES-mediated translation,although the nature of such factors remain to be determined(23). Recent analyses of HAV IRES function indicate a rolefor PCBP2 in HAV translation (162). PCBP2 was found tobind specifically to within the polypyrimidine tract of the HAVIRES, corresponding to HAV nt 1 to 157. In these experi-ments, affinity column depletion of PCBP2 from HeLa extractsresulted in only low levels of HAV translation while translationwas restored by adding back recombinant PCBP2 to the de-pleted extracts. PCBP2 has also been implicated in stimulatingpoliovirus translation (35), where it has been shown to bind to

stem-loop IV of the poliovirus IRES (34). An essential role forPCBP2 in HAV and poliovirus translation awaits further stud-ies. However, like La and PTB, it appears that PCBP2 may playa role in maintaining IRES structure in a translationally com-petent conformation. HAV RNAs lacking the 59-terminal 138nt, which are not part of the HAV IRES but do include a majorregion of the PCBP2-binding site, retained translational effi-ciency independent of PCBP2 (162). This suggests that dele-tion of the first 138 nt of the HAV RNA may mimic the effectsof PCBP2 binding and allow the IRES to spontaneously adoptthe correct structure needed to promote translation. Consid-ering that the picornavirus genomic RNA must serve as tem-plate for both translation and transcription, one may proposethat at least one role for IRES-binding proteins like La, PTB,and PCBP2 is to functionally differentiate between translationand transcription by making the RNA accessible to the trans-lational machinery. By this model, these and other proteinsmay bind to their cognate motif(s) within the IRES to induceand/or stabilize the specific RNA tertiary structures that pro-mote mRNA translation over genome transcription. On theother hand, modification, or masking, of IRES-binding pro-teins by viral and/or cellular factors may provide the signalspromoting the switch from translation to transcription.

Recent studies indicate that the IRES and IRES-bindingproteins may not act alone to promote cap-independent viralmRNA translation. In addition to an IRES, the HCV genomecontains a highly structured 98-nt 39 UTR (32, 265). Sequenceanalyses revealed that the HCV 39 UTR is highly conservedamong viral isolates, suggesting that it may function as a req-uisite element in HCV replication. This region of the HCVgenome contains a high-affinity PTB-binding site (216, 464).Interestingly, Lai and colleagues found that the presence ofthis PTB-binding site actually relieved translational repressionconferred by PTB binding to a second high-affinity site locatedwithin the HCV IRES (217, 218). It has been proposed thatPTB may control IRES-mediated translation by interactingwith both the viral RNA and an unknown factor(s) to (i) bindthe viral 39 UTR and relieve translational repression fromwithin the HCV IRES and (ii) enhance translation throughcircularization of the HCV RNA into a closed-loop translationcomplex. The exact role of PTB-mediated translational sup-pression and stimulation in the context of HCV replicationhave yet to be determined. One possibility is that PTB inter-actions with discrete regions of the HCV genome ensure theefficient translation of only genome-length RNA, and providetranslational fidelity to HCV polyprotein synthesis.

Another 39 UTR-directed mechanism of stimulating viralmRNA translation has been described in rotavirus-infectedcells. In contrast to cellular mRNAs, rotavirus mRNAs lack apoly(A) tail. Viral mRNA stability is achieved in part throughthe actions of the viral NSP3A protein, which binds to the 39end of virus-encoded mRNAs. During rotavirus infection,NSP3A directly interacts with the eIF4G isoform, eIF4GI (376).Interestingly, NSP3A may represent a virus-specific analog ofPABP, in that it appears to compete with PABP for interactionwith eIF4G and participation in the translation initiation pro-cess. Results from in vitro experiments suggest that NSP3Amay actually “evict” PABP from the eIF4F translation initia-tion complex. These results support a model in which NSP3Amediates selective translation of viral mRNA by (i) disruptingthe cellular mRNA translation through “eviction” of PABPfrom the “closed-loop” translation complex and (ii) facilitatingviral mRNA translation through interactions with eIF4G andthe viral mRNA 39 end. Moreover, eviction of PABP from thetranslation complex is likely to contribute to host protein syn-



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thesis shutoff during rotavirus infection by reducing the overallefficiency of host mRNA translation.

Influenza virus. Similar to the previous examples providedby the picornaviruses, influenza virus depends on the recruit-ment of specific host factors to mediate selective and efficienttranslation of viral mRNAs during infection. Influenza virus isresponsible for up to 70,000 deaths a year in the United Statesand 20,000,000 worldwide in the worst epidemic years andremains one of the most dreadful threats for a recurring viruspandemic. Like other cytopathic viruses, influenza virus dra-matically perturbs the normal synthesis of host macromole-cules. However, unlike most nononcogenic RNA viruses, thereplicative cycle of influenza virus includes both a nuclear andcytoplasmic phases. Accordingly, the evolution of the transla-tional strategies of this virus is likely to be dictated by a dif-ferent set of selective pressures (274). Given its small negative-strand, segmented RNA genome size, it is almost intuitive thatsuch a successful virus must have evolved a plethora of inge-nious schemes to ensure selective and efficient translation ofviral mRNAs. In the sections that immediately follow, we re-view our present understanding of how influenza virus imposesselective translation of viral mRNAs over cellular mRNAs, butyet manages to keep the infected cells translationally compe-tent, such that the steps involved in the protein synthetic path-way are not compromised.

Selective translation of influenza virus mRNAs. The mech-anisms by which influenza virus mediates selective translationof viral mRNAs are summarized in Table 3. In influenza virus-infected cells, cellular mRNAs are subjected to a modest de-crease in transcription rates (approximately twofold) (250,251). In addition, degradation of cellular mRNAs is evidentlate after infection (22, 215). Moreover, newly synthesizedcellular mRNAs fail to reach the cytoplasm after infection duethe degradation of nuclear RNA early after infection (250).Although the mechanism has not been determined, it is thoughtthat influenza virus-induced cellular mRNA degradation in thenucleus is initiated by the cleavage of the 59 ends of cellularRNA polymerase II transcripts by the viral cap-dependentendonuclease. The decapped RNAs would probably be moresusceptible to degradation by cellular nucleases, since it is wellestablished that the 59 cap structure stabilizes mRNAs (274).

However, the above events cannot completely account forthe dramatic cessation of cellular protein synthesis, becausepreexisting cytoplasmic cellular mRNAs are stable and func-tional when tested in cell-free translation systems (143, 250).Thus, the shutoff of cellular protein synthesis is not primarily

due to the degradation or modification of cellular mRNAs.Indeed, polyribosome analysis unveiled that there is a sig-nificant diminution in the association of cellular mRNAswith large polyribosomes (247). Furthermore, many cellularmRNAs remained polyribosome associated even though thesemRNAs were not translated inside the infected cell. Althoughthere were hints in the literature that actively translatedmRNAs were associated with a pool of polyribosomes thatwere cytoskeleton bound (298), subsequent studies showedthat the polyribosomes containing both the viral and nontrans-lated cellular mRNAs were associated with the cytoskeleton(252). This is in contrast to poliovirus-infected cells, in whichcellular mRNAs are dissociated from the polyribosomes andthe cellular cytoskeleton (252, 298). These observations sug-gest that there are other mechanisms that direct preferentialinfluenza viral protein synthesis.

Contribution of influenza virus mRNA structure upon se-lective translation. It was conceivable that translational se-lectivity, at least at the level of initiation, could be due tocompetition between cellular and viral mRNAs for limitingcomponents of the translational machinery as shown in othersystems, including the reoviruses (309, 388, 471). Since viralmRNAs do not have an advantage merely due to sheer mass(143), it was likely that the influenza virus mRNAs are intrin-sically better initiators of translation due to certain unknownstructural qualities. Such structural features could deceive thecellular protein-synthesizing machinery into making only viralproteins (141, 143). The first persuasive evidence that influenzavirus mRNAs are intrinsically efficient initiators of translationwas provided by studies in which cells were doubly infectedwith influenza virus and adenovirus (246, 247, 249). Theseearly experiments also showed that influenza virus also has astrategy to sustain overall high levels of protein synthesis.

In these adenovirus-influenza virus doubly infected cells,influenza virus proteins accumulated essentially to the samelevels as in cells infected by influenza virus alone (246). Thesedata suggested that influenza virus is able to overcome thehalts on host cell mRNA transport and translation imposed byadenovirus (14), demonstrating that the virus establishes itsown translational and transport regulatory mechanisms. Fur-thermore, influenza virus mRNAs were more efficiently trans-lated than late adenovirus mRNAs, which are believed to bestrong mRNAs due to the presence of the tripartite leadersequences (310). Further evidence that the structure of influ-enza virus mRNAs plays a key role in their selective translationwas obtained from a study conducted by Alonso-Caplen et al.(5). The authors found that the translation rate of the influenzavirus nucleocapsid protein (NP) mRNA, expressed from a re-combinant adenovirus, was equivalent to that of the nativeNP expressed by influenza virus itself. The recombinant NPmRNA translation rates were remarkably efficient despite theabsence of all other influenza virus gene products. Thus, thesestudies indicated that the sequence and/or structure alone ofan influenza virus RNA can confer enhanced translatability.

Because influenza virus is not readily amenable to geneticstudies, researchers have resorted to alternative strategies tostudy the contribution of the viral mRNA structure to selectivetranslation. Definitive evidence that influenza virus mRNAshave an innate ability to be preferentially translated was ob-tained from transfection and infection studies in which repre-sentative viral or cellular cDNAs were transfected into COS-1cells, which were then infected with influenza virus (140). Itwas shown that mRNA translation, directed by cellular trans-fected genes such as interleukin-2 or secreted embryonic alka-line phosphatase (SEAP) was markedly shut off after viralinfection. In contrast, an exogenously introduced influenza vi-

TABLE 3. Translational regulatory mechanisms of influenza virus

Strategy Reference(s)

Host shutoff of cellular protein synthesis .................250, 288Inhibition of cellular mRNA transport and

degradation of mRNAs in the nucleus .................250Inhibition of PKR by P58IPK ......................................135, 249, 294–296, 447Inhibition of PKR by NS1 ..........................................184, 312, 448Cap-dependent, selective translation of

influenza virus mRNAs ...........................................140, 142, 247, 250, 362Inhibition of cellular mRNA translation at

initiation and elongation stages .............................140, 247Innate ability of influenza virus mRNAs for

preferential translation............................................140, 142, 247Viral 59 UTR-mediated preferential trans-

lation of influenza virus mRNAs ...........................361, 362Recruitment of eIF4G to the viral mRNA ..............9Dephosphorylation of eIF4E......................................110Temporal regulation of influenza virus

protein synthesis.......................................................102, 486, 487



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rus gene encoding NP was not subjected to the translationalblocks imposed on the cellular genes. Subsequent studies usingchimeric constructs in which the viral 59 UTR was fused to acellular mRNA demonstrated that the selective translation ofinfluenza virus mRNAs is mediated almost exclusively by theviral 59 UTR (142).

Influenza virus recruitment of Grsf-1. Unlike the IRES ofpoliovirus or the tripartite leader of adenovirus, influenza virusmRNAs do not possess any extensive secondary structures.Instead, they contain short and relaxed 59 leaders with noupstream AUGs. Using gel mobility shift and UV cross-linkinganalysis, several factors have been identified that bind to cis-acting sequences present in the viral 59 UTR (361). A yeastthree-hybrid screen using a HeLa cell cDNA library revealedthat one of these 59 UTR-interacting factors turns out to be thecellular protein GRSF-1 (362). In vitro and in vivo bindinganalyses demonstrated that GRSF-1 can specifically bind tothe NP 59 UTR but not select NP 59 UTR mutants or cellularRNA 59UTRs (362) (Fig. 12A). Importantly, recombinantGRSF-1 was found to specifically stimulate the translation ofan NP 59 UTR-driven template in cell-free translation systems.Furthermore, the translation efficiency of NP 59 UTR-driventemplates was markedly reduced in GRSF-1-depleted HeLacell extracts but was restored by GRSF-1-in reconstituted ex-tracts. Competition experiments using NP 59 UTR sequencessimilarly demonstrated a requirement for GRSF-1 binding inthe translation of viral but not cellular mRNA (Fig. 12B).Taken together, these results demonstrate a specific interac-

tion between GRSF-1 and the influenza virus 59 UTR. Moreimportantly, these results suggest that influenza virus may re-cruit GRSF-1 to the 59 UTR to ensure the selective translationof viral mRNAs in infected cells.

It is not yet known how GRSF-1 affects the overall transla-tion efficiency of influenza virus mRNAs within the infectedcell. GRSF-1 was first cloned by probing protein blots with alabeled G-rich RNA element probe (384), which is predomi-nantly present in the cytoplasm, contains three RNA recogni-tion motifs (RRM), and belongs to the RRM protein super-family (Fig. 12C). Consistent with its RNA-binding properties,GRSF-1 also possesses a carboxyl-terminal acidic domain. Sev-eral models have now been proposed to explain how GRSF-1mediates selective translation (Fig. 13) (362). Potentially rele-vant is the observation that eIF4E, the subunit responsible forrecognition of the cap structure, is modestly dephosphorylatedin influenza virus-infected cells (110). Furthermore, influenzavirus mRNAs display exceptional ability to be translated inadenovirus-infected cells where eIF4E is severely dephosphor-ylated. eIF4E dephosphorylation leads to a decrease in thecap-binding activity (392), which may contribute to the inhibi-tion of the cellular mRNA translation in influenza virus-in-fected cells. As an RNA-binding protein, it is conceivable thatGRSF-1 may promote eIF4E function and interaction with thecomponents of the cap-binding complex to stimulate mRNAtranslation. Alternatively, GRSF-1 may directly participate inribosome recruitment to the mRNA, possibly bypassing thelimitations imposed by reduced eIF4E function. One possible

FIG. 12. GRSF-1 binds the 59 UTR of influenza virus mRNAs to stimulate viral mRNA translation. (A) Specific interaction of GRSF-1 with the 59 UTR of influenzavirus NP mRNA. The 59 UTR of the viral NP mRNA has been functionally divided into at least three contiguous sequence elements (denoted by A to C) (361). The12-nt A region (underlined) is conserved in all influenza virus mRNAs (274). Dashed lines indicate deleted regions within the respective 59 UTR constructs used toassess GRSF-1 binding. AINV refers to the NP 59 UTR in which the sequence of the A region has been inverted. The sequence of a control 59 UTR from the cellularSEAP gene is shown at the bottom. The A and B regions of the NP 59 UTR are both required for binding to GRSF-1 (362). (B) Competition for GRSF-1 selectivelyblocks translation from the NP 59 UTR in a cell-free system. To determine the contribution of GRSF-1 to influenza virus mRNA translation, the expression of aluciferase reporter protein was placed under control of the NP 59 UTR. Relative luciferase activity from the resulting NP-Luc construct was assessed in a cell-free systemin the absence (No comp) or presence of competitor oligonucleotides encoding the NP 59 UTR (NP), NP-A, SEAP, or NP-C. (C) Structural representation of GRSF-1.The three RRMs are indicated in black. The 41-amino-acid acidic domain is shown in gray.



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scenario is that GRSF-1 participates in the formation of theRNA-binding complex with eIF4G and eIF4A, independent ofeIF4E (Fig. 13). In either case, eIF4G is likely to be required,since influenza virus mRNA translation is acutely cap depen-dent (244, 274).

Temporal regulation of influenza virus mRNA translation.In addition to regulating cellular mRNA translation, influenzavirus carries out temporal regulation of its own viral geneexpression at the level of translation. For example Yamanakaet al. (486, 487) transfected HeLa cells with CAT reporter geneappended to the 59 UTR of each of the viral mRNAs sepa-rately and superinfected these cells with influenza virus. Theymeasured CAT activity at early and late times after infection.When the CAT construct contained the 59 UTR of an mRNAencoding an early protein such as NS, CAT activity was in-creased early after infection. Conversely when this was donewith a late viral protein such as neuraminidase, CAT activitywas higher at late times after infection. This avenue of inves-tigation has not been pursued further, and exact sequences ortrans-acting factors responsible for this regulation have notbeen identified. More recently, however, it was found that theinfluenza virus NS1 protein could stimulate the synthesis ofthe viral M1 protein (102). Site-directed mutagenesis stud-ies showed that specific sequences within the M1 mRNA 59UTR are required for this stimulation. These data, taken to-gether with results discussed above, suggest a key role for the59 UTR of influenza virus mRNAs in dictating temporal andselective translation in the infected cell.

Maintenance of translation in influenza virus-infected cells.Not only does influenza virus have to exert preferential trans-lation of its viral mRNAs, but also it has to ensure that the cellremains optimally translationally competent during infection(244). Without both these major strategies, viral replicationmight be compromised, a scenario unacceptable to an activelyreplicating cytopathic virus. The translational competence ofthe infected cell is assured because influenza virus has devel-oped an intricate strategy to repress the activity of the PKRprotein kinase. That influenza virus represses PKR was firstdemonstrated by analyzing cells doubly infected with influenza

virus and the adenovirus VA RNAI-negative mutant dl331(246, 247). In cells infected by dl331 alone, there was a dra-matic decline in the levels of both viral and cellular proteinsynthesis (458). This was due to excessive phosphorylation ofthe eIF2a subunit by an active PKR, which cannot be inhibiteddue to the absence of the virus-encoded VA RNAI (reviewedby Mathews and Shenk [323]). In contrast, when dl331-infectedcells were superinfected with influenza virus, a reduction of theprotein kinase activity normally detected during dl331 infec-tion was observed (246, 247). These data provided the firstevidence that influenza virus encodes or activates a gene prod-uct that, analogous to VA RNAI, inhibits PKR and preventsany resultant inhibition of protein synthesis initiation. It wassubsequently shown that the suppression of PKR activity alsooccurs in cells infected by influenza virus alone (253).

Recruitment of P58IPK and inhibition of eIF2a phosphory-lation during influenza virus infection. A number of eukary-otic viruses have devised one or more strategies to minimizethe deleterious effects on protein synthesis caused by activationof PKR (for a recent review, see reference 131). Unlike thestrategies used by other viruses described above, influenza vi-rus utilizes at least two strategies, involving a cellular and aviral protein, to block PKR activity. During viral infection,influenza virus mobilizes a cellular protein, termed P58IPK, torepress PKR activity by blocking both the autophosphorylationof PKR and the subsequent phosphorylation of eIF2a by anactive form of PKR (294, 295). In uninfected cells, P58IPK ap-pears to form an inactive complex with its own inhibitor,termed I-P58IPK (296). In response to activating stimuli, suchas viral infection or other cellular stresses, P58IPK is releasedfrom its inhibitor. As a result, PKR activity is repressed by aphysical interaction between P58IPK and PKR (130, 135, 378).To further complicate the story, two different inhibitors ofP58IPK, the molecular chaperone Hsp40 (332) and anothercellular protein P52IPK (128), have recently been identified. Ithas been postulated that these independent P58IPK complexesare regulated by distinct cellular pathways (128). Once re-leased from its inhibitor, P58IPK may negatively regulate PKRactivity through multiple steps, including the recruitment of

FIG. 13. Models for the selective translation of influenza virus mRNAs by GRSF-1. GRSF-1 binding may be facilitated by the AGGGU sequence spanning thejunction of the A and B regions within the NP 59 UTR (362). The two models shown reflect the cap dependency and requirement for eIF4G in influenza virus mRNAtranslation. (I) GRSF-1 may participate in recruitment of the cap-binding complex to the mRNA by physically interacting with one or more components of eIF4F.Active recruitment of the eIF4F complex by GRSF-1 may overcome the reduced affinity of phosphorylated eIF4E for the m7G cap that occurs during influenza virusinfection (110). As a result, GRSF-1 may enhance ribosome binding to the mRNA by increasing the stability of the eIF4F-mRNA complex (bottom). (II) Alternatively,GRSF-1 may allow viral mRNA translation to proceed independently of eIF4E by directly participating in ribosome recruitment with the other components of the eIF4Fcomplex. In this model, GRSF-1 would function in the absence of eIF4E to promote ribosome binding to the mRNA (bottom), thereby avoiding the limitations ontranslation due to eIF4E phosphorylation. 4E, eIF4E; 4G, eIF4G, 4A, eIF4A.



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molecular chaperone Hsp70 to inhibit kinase activity (333) anddisruption of PKR dimerization (447).

Role of the influenza virus NS1 protein in viral mRNAtranslation. The NS1 protein of influenza virus is an RNA-binding factor that inhibits both the nuclear export of poly(A)-containing mRNA and the splicing of pre-mRNA (86, 119, 311,385). In addition to binding to poly(A) mRNA and a stem-bulge region in U6 small nuclear RNA, the NS1 protein bindsto dsRNA (183). Evidence has accumulated to indicate thatNS1 is involved in the translation of select viral mRNAs, in-cluding those encoding the viral matrix and nucleocapsid pro-teins (361). In this regard, NS1 is thought to interact with theviral 59 UTR to selectively stimulate the initiation of viralmRNA translation (244). To shed some light on how suchtranslational selectivity may occur, recent evidence now indi-cates that NS1 can also form a complex with eIF4G in extractsfrom influenza virus-infected cells (T. Aragon, S. de la Luna,I. Novoa, L. Carrasco, J. Ortin, and A. Nieto, submitted forpublication). Thus, we may hypothesize that NS1 can recruiteIF4G to the viral 59 UTR to facilitate translation initiation.Such an interaction could also contribute to the host shutoffdue to competition with cellular mRNAs for eIF4G. Clearly,the exact mechanisms of NS1 specificity in mediating selec-tive viral mRNA translation remain to be determined. It willbe interesting to examine whether NS1 may interact withGRSF-1 to synergistically promote selective viral mRNAtranslation.

NS1 may also function during influenza virus infection toblock the actions of PKR. Given the RNA-binding propertiesof NS1 and its ability to bind dsRNA, it was hypothesized thatNS1 could compete with PKR in influenza virus-infected cellsfor binding to dsRNA. This tenet is supported by the observa-tion that NS1 inhibits the activation of PKR and, as a result,the phosphorylation of eIF2a in vitro (312). Furthermore, theNS1 protein also blocks the inhibition of translation caused bydsRNA-mediated activation of PKR in reticulocyte lysate ex-tracts. The relevant role of NS1 in PKR regulation is furtherstrengthened by the finding that the two proteins can form aspecific complex in vitro (448). An inactive mutant of NS1,which lacks a functional RNA-binding domain, was unable tobind to PKR. Moreover, a PKR mutant defective in dsRNAbinding did not interact with or inhibit the NS1 protein in vivo.These results suggest that NS1 exerts its effect, at least in part,

through heterodimerization with PKR, possibly in an RNA-dependent manner. However, discretion is advised when usingdsRNA-binding mutants of PKR to determine the mechanismof these interactions because both the dsRNA-binding andprotein interaction properties of PKR are closely embedded inthe same regions. At any rate, the fact that NS1 plays an in vivorole in modulating PKR function was recently demonstrated bystudies of mutant influenza viruses with a defective NS1 pro-tein (184). These variant viruses could not block the activationof PKR in infected cells, leading to enhanced phosphorylationof eIF2a and suppression of mRNA translation. Furthermore,the level of phosphorylation of PKR and eIF2a was well cor-related with the defect in virus protein synthesis. Consistentwith its role in translation modulation, the NS1 protein hasbeen shown to stimulate the translation of viral mRNAs (86,102), although it has not been determined if the PKR pathwayis involved. Collectively, these results suggest that NS1 mayfacilitate viral replication by blocking the critical PKR-depen-dent arm of the cellular IFN response. This idea is supportedby work by Garcia-Sastre et al., who used reverse genetics toengineer a recombinant influenza virus, termed delNS1, whichlacks the NS1 gene (139). Similar to wild-type influenza virus,delNS1 replicated to high titer within cells deficient in IFNsignaling pathways. However, delNS1 replication was severelylimited in cells in which IFN signaling remained intact. Simi-larly, delNS1 replicated to lethal titers in mice with a targetdeletion in the STAT1 gene, which renders cells unable torespond to IFN (77, 334). IFN-competent control mice effec-tively suppressed delNS1 replication. Thus, the NS1 gene ofinfluenza virus may not play a direct role in viral replication,but, rather, it functions to block the antiviral effects of the hostIFN system. In this regard, NS1 may confer translational com-petence to influenza virus by removing the translational block-ade imposed by PKR.

Influenza virus appears to encode more than one strategy torepress PKR function (Table 4). Influenza virus is known togenerate large amounts of both negative-strand and positive-strand viral RNAs during infection, forming dsRNAs that arecapable of activating PKR. It seems logical that the virus usesa cellular factor (P58IPK) and a viral protein (NS1) to inhibitthe activation of PKR in order to ensure efficient protectionagainst the resulting inhibition of translation that would blockvirus replication. Alternatively, some of these PKR inhibitors

TABLE 4. Viral mechanisms of PKR inhibition

Target Virus/inhibitor Mechanism of action Reference(s)

dsRBMs Adenovirus/VAI RNA Competitively inhibits dsRNA-binding reaction 320, 321Epstein-Barr virus/EBER RNAs Competitively inhibits dsRNA-binding reaction 64, 70Vaccinia virus/E3L Sequesters dsRNA 55, 475Influenza virus/NS1 Sequesters dsRNA 312Reovirus/s3 Sequesters dsRNA 20, 307, 493HIV TAR RNA Competitively inhibits activator dsRNA binding; may activate

PKR; could target TAR RNA binding protein to PKR169, 314

TAR RNA binding protein Binds to PKR, inhibits kinase activity; may sequester dsRNA 24, 73

Dimerization Influenza virus/P58IPK Disrupts dimerization 294Hepatitis C virus/NS5A Disrupts dimerization 129, 133, 134

Substrate interaction Vaccinia virus/K3L Binds to PKR as a pseudosubstrate; blocks PKR-substrate in-teraction

21, 255HIV Tat 39, 327

Expression and stability Poliovirus Induces PKR proteolysis 29, 30HIV Tat Lowers expression of PKR 397

Regulation of eIF2a HSV/g134.5 gene product Complexes with eIF2a and induces an eIF2a phosphatase 188, 338Simian virus 40/T antigen Targets processes downstream of eIF2a 386, 444



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may interfere with activities of PKR not directly related to theregulation of protein synthesis. Perhaps such viral multistrat-egies are designed to fine-tune the activities of the enzyme atdifferent stages in the viral life cycle. Furthermore, since PKRis found in both the nucleus and cytoplasm (68) the use of NS1,a predominantly nuclear protein (166), and P58IPK, a cytoplas-mic protein (268), may allow influenza virus to control PKRfunctions in both cellular compartments. Several key questionsremain to be addressed in future studies. (i) How does influ-enza virus activate the P58IPK-PKR regulatory pathway? (ii)Would cells devoid of P58IPK be more susceptible to influenzavirus infection and replication? (iii) Do NS1 and P58IPK workin a synergistic manner to inhibit PKR function and enhancethe translation of viral mRNAs?

VIRAL MODIFICATION OF CELLULAR FACTORS

Viral replication requires a large amount of energy andthereby demands almost total metabolic control of cellularresources. As discussed in the previous sections, the competi-tion for resources imposed by these conditions has created aplaying field in which viruses have evolved mechanisms tosupersede cellular mRNA translation through the recruitmentand/or modification of translation factor function. Many ofthese processes of translation factor modification may havearisen through the efforts by viruses to block cellular counter-measures aimed at disrupting viral mRNA translation. As de-scribed in this section, the effects of translation factor modifi-cation range from altering the efficiency of cap-dependenttranslation and translation elongation to altering the rate ofglobal mRNA translation and the efficacy of the innate antivi-ral response of the host cell.

Inactivation of eIF4E and modulation of the eIF4E-bindingproteins. eIF4E may be considered the pivotal translation ini-tiation factor. Compared to the other translation factors, it ispresent in limiting amounts in the cell, where it is required forassembly of the 59 cap-binding complex. As described above,eF4E binds to the 59 ends of both cellular and viral mRNAsand interacts with eIF4A and eIF4G. This macromolecularcomplex constitutes eIF4F and facilitates the binding of themRNA to the 43S preinitiation complex (335). The affinity ofeIF4E for the mRNA 59 cap is increased by phosphorylationon serine 209 and occurs in response to mitogenic stimulation(237, 431, 432). Analyses of eIF4E activity in the presence orabsence of serine 209 phosphorylation has indicated thatphospho-eIF4E is stimulatory for mRNA translation (432).The cellular enzyme responsible for serine 209 phosphoryla-tion has been putatively identified as the Mnk1 protein kinase.Mnk1 was shown to phosphorylate eIF4E on serine 209 in vitro(473). More recent studies demonstrated that overexpressionof Mnk1 could induce high levels of eIF4E phosphorylationin vivo (474), although the effects of Mnk1 overexpressionon mRNA translation were not directly examined. These re-sults are consistent with previous observations demonstrat-ing that mitogen-induced eIF4E phosphorylation was depen-dent on the activity of the extracellular signal-related kinaseand mitogen-activated protein kinase (339, 341, 472). Thus,Mnk1 may link eIF4E phosphorylation and translation stimu-lation through the mitogen-activated protein kinase-signalingpathway (387). In general, eF4E function is a requisite forcap-dependent translation but is thought to play a more criticalrole in the translation of mRNAs that have long 59 UTRs withregions of extensive secondary structure that are nonconduciveto ribosomal scanning. These mRNAs are translated ineffi-ciently due to limitations in eIF4E and the associated helicaseactivity of the assembled eIF4F complex (reviewed by Sonen-

berg [432]). Serine 209 phosphorylation is thought to increasethe translational efficiency of these mRNAs by stimulatingeIF4E cap-binding activity and increasing the level of mRNA-bound eIF4F helicase activity sufficiently to melt mRNA sec-ondary structure.

The activity of eIF4E is also regulated through direct inter-action with the eIF4E-binding proteins, 4E-BP1 to 4E-BP3(367, 380). The 4E-BPs are low-molecular-weight proteins thatlink cap-dependent translation to mitogenic signaling pathwaysand are themselves directly regulated by phosphorylation (105,305, 367). A model for eIF4E regulation by the 4E-BPs hasbeen presented (387, 432). This model is based on the obser-vations that interaction of eIF4E with 4E-BP1 disrupted theassembly of an eIF4E-eIF4G complex (172). 4E-BPs may fa-cilitate the suppression of cap-dependent translation by bind-ing to eIF4E and preventing the formation of an active eIF4Fcomplex. Phosphorylation of the 4E-BP results in dissociationof the eIF4E–4E-BP inhibitory complex (367), thereby stimu-lating mRNA translation (387). 4E-BP phosphorylation andtranslation stimulation occur in response to mitogenic signal-ing and other cell growth-modulatory stimuli (154, 469). Elu-cidation of such cellular signaling cascades is currently an in-tense area of research. Viral disruption of eIF4E regulationmay therefore have far-reaching consequences beyond support-ing viral replication, including implications for apoptosis andoncogenic transformation (432).

Viruses have targeted the processes of eIF4E regulation tofacilitate translational selectivity for viral mRNAs during in-fection. In mammalian cells, the level of eIF4E serine 209phosphorylation is reduced during infection with a number ofviruses, including adenovirus and influenza virus (110, 244,411, 499). In short, the block in eIF4E phosphorylation corre-sponds to a decrease in the level of host mRNA translationwith little or no effects on translation of viral mRNAs. Themolecular mechanisms by which eIF4E phosphorylation is re-duced by influenza virus are not clear. Examination of mRNAtranslation in cells infected with influenza virus revealed that(i) virus infection significantly reduced the extent of eIF4Ephosphorylation and the pool of active eIF4E and (ii) theefficiency of viral mRNA translation was insensitive to theresulting low levels of functional eIF4E and the eIF4F complex(110). All influenza virus mRNAs contain a short, conserved 59UTR predicted to possess limited, if any, secondary structure(274). This short linear 59 UTR directs viral mRNA translationwith a remarkably high efficiency. Such “strong” mRNAs maybe less sensitive to limitations in the availability of eIF4F andassociated helicase activity imposed by eIF4E dephosphoryla-tion. As a result, dephosphorylation of eIF4E during influenzavirus infection may favor the translation of viral mRNAs overthe host mRNA and therefore may contribute to the hostshutoff phenomenon. At this point, it is not known if influenzavirus induces an eIF4E phosphatase activity, inhibits an eIF4Ekinase including possibly Mnk1, or simply makes eIF4E un-available for phosphorylation. These possibilities should pro-vide a fertile area for future research in viral translationalcontrol mechanisms.

In cells infected with adenovirus, the block in eIF4E phos-phorylation plays a dual role of contributing to the host shutoffduring late-stage infection and selecting for the translation (byribosome shunt) of viral mRNAs that contain the tripartiteleader. The actual mechanism by which adenovirus preventseIF4E phosphorylation is not clear. Evidence from experi-ments that examined infection kinetics and levels of eIF4Ephosphorylation has implicated a late adenovirus gene func-tion in the coordinate reduction in eIF4E phosphorylation(499). An attractive hypothesis is that adenovirus may directly



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or indirectly inhibit Mnk1 or another cellular protein kinase(s)that phosphorylates eIF4E.

Viral regulation of eIF4E function also occurs at the level of4E-BP activity. Both poliovirus and EMCV inhibit 4E-BP phos-phorylation within infected cells (155). Inhibition of 4E-BPphosphorylation may therefore contribute to the host shutoffof protein synthesis observed during picornavirus infection.This idea is supported by analyses of 2A-pro deficient strainsof EMCV. Loss of 2A-pro decreased the efficiency of viralprotein synthesis and abolished virus-induced host shutoff ininfected cells (441). Interestingly, mutant viruses exhibitedenhanced viral replication and increased efficiency of viralmRNA translation during infection in the presence of rapa-mycin and wortmannin, chemical inhibitors of 4E-BP phos-phorylation (25). Thus, inhibition of 4E-BP phosphorylationcomplements EMCV mutations in 2A-pro to rescue viralmRNA translation (441). Together, these results indicate thatinhibition of 4E-BP and repression of eIF4E function contrib-utes to the host shutoff induced by picornavirus infection (26).

Cleavage of eIF4G. With the exception of HAV, the hostshutoff induced during picornavirus infection is extremely in-tense and results in nearly complete disruption of cellularmRNA translation to favor IRES-mediated translation of theviral mRNA. A main feature of this host shutoff involves dis-ruption of eIF4F function through virus-mediated cleavage ofeIF4G (Fig. 5), although it is now clear that other factors,including inhibition of 4E-BP phosphorylation, play a role inthe shutoff process (174, 468). Cleavage of eIF4G proceeds inpart through the actions of the virus-encoded 2A-pro duringEMCV and poliovirus infections or through the actions ofprotease-L in foot-and-mouth disease virus infection (reviewedin references 197, 258, and 340). Cleavage of eIF4G effective-ly selects for cap-independent, IRES-mediated viral mRNAtranslation by removing the competition for the translationalmachinery imposed by cap-dependent cellular mRNA transla-tion.

The implications of eIF4G cleavage upon host cap-depen-dent mRNA translation are best understood by examiningeIF4G structure and function. eIF4G has been cloned fromseveral different species and is present as structurally distinctisoforms that contain binding sites for interaction with eIF4E,eIF4A, eIF3, PABP, and the Mnk1 protein kinase (153, 214,258, 340). The current model for eIF4F function in cap-depen-dent translation proposes that eIF4G serves as a molecularbridge for the assembly of the cap-binding complex upon the 59end of the mRNA and facilitates its interaction with the 43Spreinitiation complex. Furthermore, eIF4G may potentiateeIF4E phosphorylation and increased eIF4E activity by re-cruiting Mnk1 to the eIF4F complex (153, 383, 474). Poliovirus2A-Pro cleaves eIF4G to yield discrete amino- and carboxyl-terminal cleavage products (Fig. 14). This prevents assembly ofa functional eIF4F complex, thereby disrupting an essentialstep in cap-dependent translation initiation. Interestingly, cap-

independent viral protein synthesis of select picornaviruses isstimulated by the carboxyl-terminal eIF4G cleavage product,which contains the binding sites for eIF3 and eIF4A (340, 354).

Recent evidence indicates that eIF4G isoforms are differen-tially targeted and cleaved upon picornavirus infection. Thehost shutoff of protein synthesis during poliovirus infection hasbeen attributed to cleavage of eIF4GII. Importantly, eIF4GIIcleavage was shown to coincide with the kinetics of host shutoffof protein synthesis and occurred during later-stage infec-tion, after the cleavage of eIF4GI (161). Similarly, cleavageof eIF4GII was shown to occur during later-stage human rhi-novirus infection, again coinciding with the kinetics of the hostprotein synthesis shutoff (440). Together, these results demon-strate that the different eIF4G isoforms are differentially tar-geted during picornavirus infections and suggest that eIF4GIIcleavage may be the rate-limiting step in the shutoff of hostprotein synthesis during poliovirus and rhinovirus infection.

Cleavage of PABP: disruption of the closed-loop translationcomplex. In addition to cleavage of eIF4E, picornaviruses maydisrupt host mRNA translation through the modulation ormodification of PABP. PABP is a 70-kDa RNA-binding pro-tein that has remained highly conserved in evolution and playsa direct role in mRNA stability through its interaction with thepoly(A) tails of mRNA (225). Recent evidence indicates thatPABP directly participates in mRNA translation by function-ing to bring the 39 UTR of the mRNA into the proximity of the59 cap and the cap-binding complex. As described above, thiscircularization of the translation initiation complex is facili-tated through PABP interactions with the mRNA poly(A) tailand eIF4G. The resulting “closed-loop” translation initiationcomplex is thought to stabilize assembled initiation factors andincrease translation efficiency (225, 401). Thus, viral disruptionof PABP function can be expected to (i) alter mRNA stabilityand (ii) reduce the overall rate and efficiency of cap-dependentmRNA translation.

Interestingly, it now appears that PABP is targeted for cleav-age by 2A-Pro of the enteroviruses, coxsackievirus, and polio-virus. Analysis of PABP during coxsackievirus infection re-vealed that it was proteolytically cleaved during infection(259). In these studies, PABP was efficiently cleaved in vitroand in vivo by the virus-encoded 2A-Pro. Cleavage of humanPABP occurred in a unique position that resulted in separationof the RNA-binding activity from the homodimerization activ-ity. Importantly, the proteolytic fragments were inefficient atstimulating mRNA translation, suggesting that PABP cleavagemay impart host translational suppression. Similar observa-tions were extended to poliovirus infection, in which PABPcleavage was shown to occur through the actions of the viral2A-Pro and, to a lesser extent, the viral 3C protease (233). Inboth studies PABP cleavage was accompanied by a dramaticloss of cellular translational activity in vitro and in vivo (233,259). Taken together, these results suggest that direct cleavageof PABP may contribute to the inhibition of host mRNAtranslation during enterovirus infection. However, the extentto which PABP cleavage contributes to host protein synthesisshutoff, versus cleavage of eIF4G, has not been directly com-pared. Moreover, using infected cells, it will now be importantto separate the effects of PABP cleavage on mRNA stabilityfrom direct translation-regulatory effects due to disruption ofthe “closed-loop” translation complex. It is tempting to pro-pose that the cleavages of the PABP and eIF4G isoforms occurat distinct stages during viral infection. In this case, PABPcleavage may function to disrupt ongoing host translation whileeIF4G cleavage events may be directed toward blocking denovo cap-dependent translation during infection.

FIG. 14. Domain structure of eIF4G. The arrow points to the site of cleavageby poliovirus 2A-Pro. Shaded areas indicate the eIF4E-, eIF3-, and eIF4A-binding domains. The bar indicates the region responsible for binding to Mnk1.Numbering refers to the prototypic eIF4GI (153).



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Modification of EF-1. In addition to modifying the compo-nents necessary for translation initiation, viruses may targetthe process of translation elongation to facilitate the efficienttranslation of viral mRNA. Translation factor EF-1 catalyzesthe critical step of delivering the aminoacyl-tRNAs to the elon-gating ribosome. The viral regulatory protein, ICP0, forms astable complex with EF-1d during HSV infection (256). Anal-ysis of the translation efficiency of a reporter protein in RRLrevealed that the addition of recombinant ICP0 repressedtranslation in a dose-dependent fashion. Although the actualrates of translation elongation and the efficiency of translationinitiation were not addressed, it was concluded that ICP0 mayfunction during specific stages of HSV infection to modulatethe translation of cellular and viral mRNAs. Interestingly,EF-1d exists as hypo- and hyperphosphorylated isoforms with-in HSV-infected cells (256). Analysis of HSV mutants suggeststhat EF-1d phosphorylation is mediated by a protein kinase en-coded by the product of the viral U(L)13 gene (257). Althougha physiological role for U(L)13-mediated phosphorylation ofeEF-1d during HSV infections has not been demonstrated, it isinteresting to speculate that EF-1d phosphorylation may pro-mote viral protein synthesis by inducing the coordinate disso-ciation of EF-1d from ICP0 at a specific point(s) during viralreplication. Finally, EF-1d has also been shown to interact withthe Tat protein of HIV-1 (485). In this case, the Tat–EF-1dinteraction resulted in a dramatic reduction in the efficiency ofcellular but not viral mRNA translation. As with HSV, how-ever, confirmation of EF-1 regulation by Tat, and its physio-logical role during HIV infection remain an open question.

Disruption of eIF2a phosphorylation. Eukaryotic cells re-spond to stress conditions, including viral infection, in part bydown-modulating the overall rate of protein synthesis. Thistranslational control response to stress occurs largely throughthe modification of eIF2. eIF2 functions to deliver the Met-tRNAi to the 40S ribosome, and this constitutes a rate-limitingstep to translation initiation when eIF2 is modified throughphosphorylation by specific cellular serine-threonine proteinkinases. Currently, at least five distinct eukaryotic proteinkinases have been identified that play a role in translationalcontrol by modulating eIF2 function; these are the HRI,PERK, PEK, and PKR enzymes and the GCN2 protein kinase(423). Known as the eIF2a protein kinase family, these en-zymes respond to specific signals to phosphorylate serine 51 ofeIF2a. Functional analyses of the eIF2a protein kinases indi-cate that each enzyme provides the cell with a unique ability tomodulate mRNA translation in response to specific cellularstresses (reviewed in references 201 and 477). For example, theHRI protein kinase is expressed in mammalian reticulocytesand mediates eIF2 a phosphorylation in response to hemedepletion (58). PERK and PEK reside on the endoplasmicreticulum, where they mediate translational control in re-sponse to endoplasmic reticulum stress (178, 419). The yeastGCN2 enzyme presents an example of both global and specificcontrol of mRNA translation by phosphorylating eIF2a in re-sponse to amino acid starvation (202). This results in the spe-cific stimulation of GCN4 translation and concomitant repres-sion of global protein synthesis (201). GCN4 stimulates aminoacid production by inducing the expression of amino acid-biosynthetic components. PKR is ubiquitously expressed inmost mammalian tissues. As described below, PKR is a com-ponent of the IFN-induced cellular antiviral response and apleiotropic mediator of extracellular signals (68). However, PKRis best known for its ability to phosphorylate eIF2a and repressmRNA translation. Phosphorylation of eIF2a on serine 51 byPKR or the other eIF2a protein kinases inhibits the guaninenucleotide exchange reaction on eIF2 (Fig. 15). The resulting

eIF2 [S51-phospho]-GDP binary complex has a higher affinityfor the eIF2B guanine nucleotide exchanger than does the non-phosphorylated eIF2 isoform. The increased affinity for eIF2Bimpedes eIF2B function and results in sequestration of eIF2Bwithin an inactive complex with eIF2 [S51-phospho]-GDP.eIF2B sequestering blocks the requisite recycling of GDP forGTP on eIF2 and prevents de novo eIF2–GTP–Met-tRNAiternary-complex formation. As a result, mRNA translationinitiation is blocked.

(i) PKR structure and function. PKR and its role in cellu-lar metabolism have been extensively reviewed (58, 66, 201,335, 404). Similar to many protein kinases, PKR is regulatedthrough a combination of transcriptional and posttranscrip-tional processes including regulatory interactions with P58IPK

(135, 294, 378). PKR, however, is unique among the proteinkinase superfamily in that it is the target for regulation byvirus-encoded inhibitory molecules (241–244, 321, 322). Struc-turally, PKR is composed of an NH2-terminal regulatory do-main and a COOH-terminal protein kinase catalytic domain(Fig. 16A) (68, 165, 336, 459). Ubiquitously expressed at lowlevels in virtually all mammalian tissues examined (15), PKR istranscriptionally induced by IFNs, which are secreted by hosttissues in response to viral infection (69) (see below). PKR israpidly activated after binding dsRNA or even single-strandedRNA species that possess regions of extensive secondary struc-ture (70, 165, 248, 254, 316, 325, 331). This clearly imposesproblems for many eukaryotic viruses, which possess dsRNAwithin the virion or produce high levels of dsRNA intermedi-ates during replication. PKR binds dsRNA via its two NH2-terminal regulatory domain-binding motifs (dsRBM) (Fig.16A) (113, 165, 224, 363, 395). As a result of these events, thekinase may undergo a conformational alteration which triggersthe catalytic activities of PKR (see reference 68 for a detailedreview of PKR structure and function). Typically, low levels ofdsRNA are required to initiate PKR activation while highlevels of dsRNA actually result in inhibition of PKR activationand suppression of kinase function (70). Once bound to activa-

FIG. 15. Translational control by eIF2a phosphorylation. eIF2 is composedof several subunits, including the alpha subunit (eIF2a), which is targeted forphosphorylation by the eIF2a protein kinases. eIF2 participates in mRNA trans-lation by delivering the Met-tRNAi to the incoming 40S ribosomal subunit in theform of an eIF2–GTP–Met-tRNAi ternary complex (3°) (335). After Met-tRNAibinding, eIF2 is released from the complex in an inactive state bound to GDP.The bound GDP is recycled for GTP by the eIF2B guanine nucleotide exchangefactor (dotted and gray lines), and the initiation process continues. PKR, PERK,PEK, HRI, and GCN2 can block the guanine nucleotide exchange reaction byphosphorylating serine 51 of eIF2a. As a result, the pool of functional eIF2 isdepleted and translation arrests at the initiation stage.



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tor dsRNA, PKR autophosphorylates on several critical serineand threonine residues (453), rendering the kinase active. Re-cent evidence by Zhu et al. (503) indicates that in addition tomediating dsRNA activation of PKR, the dsRBMs function totarget PKR to the ribosome, thereby potentially providing ac-cess to the PKR substrate, eIF2a (335). Activation of PKR canalso proceed through interaction with Pact, a novel PKR-acti-vating protein that also binds dsRNA (366). However, Pactappears to activate PKR by a process that is independent ofdsRNA, which presumably involves alteration of PKR confor-mation.

Comprising the COOH-terminal region of the molecule, thePKR catalytic domain facilitates the recognition of PKR sub-strates and the phosphorylation of serine and/or threoninesubstrate residues. Characteristic of all protein kinases, thisregion of PKR presents a regulatory target for kinase inhibi-tory molecules, including protein kinase pseudosubstrates (454).Indeed, recent studies indicate that the COOH-terminal re-gion of the PKR catalytic domain mediates the substrate in-teraction and is targeted for regulation by virally encodedpseudosubstrate inhibitors (73, 74, 135, 255).

Several studies indicate that dimerization between two PKRmolecules may be required for the autophosphorylation pro-cess and subsequent catalytic activity (18, 51, 284, 357, 364,395, 460). Structure-function analyses have shown that PKRdimerization can occur through both dsRNA-dependent and-independent processes (364, 365, 395). Our laboratory hasidentified a region of PKR, amino acids 244 to 296, which canmediate the dimerization process independently of dsRNA(135, 447) (Fig. 16A). Interestingly, this region also mediates adirect interaction with a diverse set of virus-directed PKRinhibitors (133, 294) that disrupt the PKR dimerization pro-cess.

PKR clearly has other substrates in addition to eIF2a, con-

sistent with its reported roles in growth factor and calcium-mediated signal transduction (344, 435), the regulation of tran-scription (266, 277, 317, 488), and the induction of apoptosis(87, 293, 490) (reviewed by Proud [382] and Williams [481]).However, it is the phosphorylation of eIF2a by PKR that ismost frequently targeted for regulation by viruses. A diagramof the events leading to PKR activation and eIF2a phosphor-ylation is shown in Fig. 16B. IFNs, produced in response toviral infection, induce the transcriptional activation of PKR,resulting in a high level of PKR expression. During virus in-fection, the binding of virus-encoded dsRNA to PKR initiatesthe PKR activation process, which includes the aforemen-tioned dimerization and autophosphorylation events (84, 301,373). Once activated, PKR phosphorylates eIF2a to limitmRNA translation. As a result of these PKR-mediated pro-cesses, viral replication is blocked at the level of protein syn-thesis (Fig. 15 and 16). High levels of eIF2a phosphorylationlead to an antiproliferative state (19, 61, 89, 337, 395). Such asituation would be incompatible with viral infection and repli-cation, providing another reason why viruses must inhibit PKR.

(ii) Mechanisms of PKR inhibition by eukaryotic viruses.To avoid the deleterious effects on viral replication due toPKR-mediated eIF2a phosphorylation, many viruses have de-veloped successful strategies to block PKR function, thusavoiding, at least in part, the antiviral effects of IFN. As sum-marized in Table 4, viruses have employed a range of mecha-nisms to inhibit PKR function, from targeting the PKR acti-vation process to regulation of catalytic function and beyond.These include directing inhibitors that (i) interfere with thedsRNA-mediated activation of PKR, usually by binding to theconserved dsRNA-binding domains or sequestering RNA ac-tivators; (ii) interfere with kinase dimerization; (iii) block thekinase catalytic site and PKR-substrate interactions; and (iv)alter the physical levels of PKR; and (v) may regulate eIF2a

FIG. 16. Structure and activation of PKR. (A) Domain structure of PKR and sites of viral regulation. The PKR regulatory domain spans amino acids 1 to 264 andincludes two dsRBMs (indicated in black) which mediate binding to activator dsRNA. The protein kinase catalytic domain comprises the PKR C terminus (amino acids265 to 551) and contains the 11 subdomains (denoted by Roman numerals) conserved in all eukaryotic protein kinases (176). Bars indicate the regions that participatein dsRNA binding, dimerization, and substrate interaction. Virus-directed inhibitors that target PKR function are listed beneath their specific target sites and arereferenced in Table 4. (B) PKR activation. PKR is transcriptionally induced by IFNs and becomes active through a process of dsRNA binding and dimerization. Onceactive, PKR can phosphorylate eIF2a. Within a virus-infected cell, PKR-mediated phosphorylation of eIF2a results in a block in protein synthesis, cell growth arrest,and inhibition of viral replication (241, 242). Additionally, PKR may participate in the regulation of other IFN-induced genes by signaling the phosphorylation of IkBor the activation of IRF-1 (277).



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phosphorylation directly, or affect components downstreamfrom eIF2a. Each PKR-inhibitory group includes a diverse setof viruses, largely unrelated except for their ability to inhibitthe kinase. In addition, some viruses employ multiple strate-gies for inhibiting PKR, resulting in pleiotropic effects on PKRfunction. It is noteworthy that such a diverse range of viruseshas utilized often-limited genomic resources to target PKR forregulation. This reflects the pivotal role played by the kinasewithin the IFN-induced antiviral response of the host cell.

Disruption of the IFN-induced cellular antiviral responsethrough inhibition of PKR. Upon infecting the cell of a verte-brate host, viruses must overcome the innate antiviral responseprovided by the cellular IFN system. IFNs are a family ofcytokines which are secreted by the cells of vertebrate animalsin response to viral infection and other cellular stresses. Onceexposed to IFN, responsive cells initiate a signaling cascadewhich culminates in the specific induction of multiple IFN-inducible genes (88), only a small subset of which have beenextensively characterized (for a review of the IFN system, thereader is directed to references 230, 346, 374, 403, 414, 415,and 437). The IFN-induced gene products, which play a role infighting virus infection, include the 29-59 oligoadenylate syn-thetase (28, 422), RNase L (421, 500), the Mx proteins (275,369), and PKR (15, 212, 336, 451, 459). As a result, IFNs directa block in viral gene expression at multiple levels, including theinhibition of viral RNA transcription and translation, and thedegradation of viral transcripts.

Paramount to the IFN-induced antiviral response is thefunction of PKR, which not only imposes a limitation on viralmRNA translation but also participates in dsRNA- and IFN-induced signaling events (Fig. 16). These functional propertiesof PKR greatly contribute to the ability of IFN to provide thebody’s first level of defense against viral infection. PKR isrequired for the induction of IRF-1 activation (261, 277),which in turn regulates the transcription of a variety of IFN-inducible genes (260), including the type 1 IFNs, IFN-a andIFN-b (277, 415, 488). PKR also contributes to the IFN-me-diated response through its ability to activate the transcriptionfactor, NF-kB (277, 297). Not surprisingly, inhibition of PKRfunction results in attenuation of the IFN response by blockingone or more of these PKR-dependent events (277, 488). Thus,PKR presents an attractive target for virus-mediated inhibitionof the host IFN response (Fig. 16).

(i) Viral inhibition of PKR: HCV. The implications stem-ming from viral inhibition of PKR and disruption of the IFNresponse are perhaps best demonstrated by very recent workon the clinically relevant HCV. HCV now infects more than2% of the worldwide population, including over 4 million inthe United States (6). HCV infection often assumes a persis-tent course, which can lead to chronic hepatitis and liver cir-rhosis, and is strongly associated with the development of hep-atocellular carcinoma and lymphoproliferative disorders (355,356, 463).

HCV infection is currently treated by parenteral administra-tion of type I IFN alone or in combination with ribiviran, anucleoside analog (326). Problematically, an increasingly highproportion of HCV-infected individuals (60 to 80%) fail torespond to IFN therapy or relapse after therapy cessation (208,213). Response to IFN therapy differs among the six HCVgenotypes but is observed, at some level, in all HCV genotypesworldwide. In an effort to understand the molecular mecha-nism(s) of HCV-mediated resistance to IFN therapy, severalresearch groups have focused attention on sequencing clinicalisolates of HCV from individuals who did or did not respondto IFN therapy (103, 104, 240, 278). What is clear from thesestudies is that sequence variation from the prototypic IFN-

resistant HCV J strain (240) within the nonstructural 5A(NS5A) protein of the HCV polyprotein cleavage product isassociated with sensitivity to IFN in Japanese HCV 1B sub-types (103, 104, 278). Viral isolates with multiple amino acidsubstitutions within a region of NS5A, termed the IFN sensi-tivity-determining region (ISDR; amino acids 2209 to 2248),were eliminated from HCV-infected patients during IFN ther-apy, while those exhibiting the prototypic ISDR sequence wereIFN resistant, persisting at therapy cessation. These resultssuggested that HCV NS5A may mediate viral sensitivity to IFNthrough specific sequences located within or around the ISDR.

Accordingly, it was demonstrated that NS5A from IFN-re-sistant strains of HCV 1A and 1B can physically bind PKR byan ISDR-dependent mechanism to inhibit kinase function, im-plicating NS5A as a mediator of the IFN-resistant HCV phe-notype (133). It was subsequently hypothesized that mutationswithin the ISDR may similarly disrupt NS5A function to ren-der HCV sensitive to the PKR-mediated antiviral effects ofIFN. Subsequent analyses revealed that ISDR sequence vari-ants of NS5A corresponding to IFN-resistant and -sensitiveclinical isolates of HCV 1B (103) exhibited differential abilitiesto control PKR function in vivo (129, 132, 134). These studiesdemonstrated that NS5A from IFN-resistant HCV disrupted acritical step of PKR activation, resulting in repression of PKRfunction. In contrast, clinically defined mutations within theISDR abrogated the PKR-regulatory function of NS5A. Fur-ther experiments mapped the PKR-interactive domain of HCVNS5A to a 64-amino-acid motif that includes the ISDR and theimmediate carboxyl-terminal flanking region (129). Thus, itnow appears that HCV may mediate resistance to IFN in partby blocking the PKR-dependent arm of the IFN response,through a direct NS5A-PKR interaction. In support of this,mutations within the PKR-binding domain on NS5A, includingthose within the ISDR, may confer IFN sensitivity by disrupt-ing the NS5A-PKR interaction. Recent work indicates this tobe true; infection of stable cell lines expressing wt or mutantNS5A with VSV revealed striking differences in the sensitivityof VSV to IFN in the presence or absence of functional NS5A(134, 377). In these studies, VSV replicated to significantlyhigh levels within IFN-treated cells expressing wt NS5A fromIFN-resistant HCV. In contrast, VSV replication was sup-pressed in cell lines expressing a nonfunctional NS5A mutant,reflecting the innate sensitivity of VSV to the antiviral effect ofIFN. Analyses of PKR function and eIF2a phosphorylationattributed the apparent rescue of VSV replication to theNS5A-mediated inhibition of PKR and a resulting block ineIF2a phosphorylation (134) (Fig. 17). Moreover, very recentevidence indicates that HCV disruption of PKR function is notrestricted to effects on eIF2a phosphorylation alone. NS5Aexpression also renders cells refractory to PKR-dependent sig-naling (134; M. Gale, Jr., unpublished data). Disruption of hostPKR signaling pathways is now considered an important mech-anism by which HCV may facilitate its domination of the hostIFN response.

VIRAL PERSISTENCE AND TRANSLATIONALCONTROL

As is clear from the material presented above, a commontheme in viral translation programming is the preferential se-lection for the translation of viral mRNAs at the expense ofhost mRNA translation. While such programming is oftencompatible with the life cycle of viruses that mediate rapid,lytic infection, such as influenza virus, poliovirus, and EMCV,it is not always compatible with viruses that mediate persistentinfection. Little is known of the nature of viral translational



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programming as it pertains to persistent infection, although itclearly requires that host mRNA translation remain sufficientto sustain the host cell and support viral persistence. In thissection, we will take a brief look at the relationship betweentranslational programming and viral persistence. As describedbelow, persistent infection may involve viral translational pro-gramming that impacts the host cell at several levels, includingthe control of translation, apoptosis, and cell growth-regula-tory pathways.

Translational programming and maintenance of viral per-sistence. Persistent infection requires the virus to ensure thatthe host cell remains translationally competent. This is impor-tant not only for the synthesis of viral proteins but also for thesynthesis of cellular proteins and continued cellular viability.Analyses of the mechanisms by which viruses may mediatepersistence and latency suggest that host cell integrity andtranslational competence are maintained through (i) viralmodulation of specific cellular mRNA translation and (ii)viral modification of host signaling and translational regulatorypathways. As described above, the continued synthesis of ribo-somal proteins during HSV-1 infection can be considered crit-ical for maintaining viral persistence and latency. Interestingly,the level of ribosomal protein S6 phosphorylation is modulatedin response to HSV-1 infection, thereby favoring the transla-tion of host 59 TOP RNAs, including those that encode theribosomal proteins (164). Modification of S6 phosphorylationmay thus facilitate translational competence, in part by ensur-ing that the synthesis of ribosomal proteins remain uncompro-mised during infection. The mechanisms by which HSV-1 in-duces the modification of S6 have not been determined. It isconceivable that viral infection results in activation of one ormore cellular protein kinases that phosphorylate S6 and/orthat a virus-encoded protein kinase may effect S6 phosphory-lation.

In addition to maintaining host translational competenceduring persistent infection, viruses must ensure that the syn-thesis of their own proteins remains uncompromised. As pre-sented above, many eukaryotic viruses encode mechanisms torepress the cellular PKR protein kinase and avoid the delete-rious effect upon protein synthesis due to high levels of eIF2aphosphorylation (131). Thus, inhibition of PKR-dependent eIF2aphosphorylation can be seen as a mechanism to ensure overalltranslational competence during viral infection. Through the

subsequent targeting of specific translational processes, includ-ing the cap-binding reaction, elongation, and termination, vi-ruses can then manipulate host mRNA translation to the over-all benefit of viral replication. However, the implications forviral disruption of PKR-dependent eIF2a phosphorylation arefar-reaching and are not limited to the effects on mRNA trans-lation. As presented above, repression of PKR provides theadded advantage of avoiding, in large part, the antiviral effectsof the host IFN response. Moreover, viral inhibition of eIF2aphosphorylation may disrupt critical host apoptotic and tumorsuppressor pathways, which rely specifically on the ability tomodify eIF2a activity (67, 68, 450).

Translational control, persistent infection, and regulationof host apoptosis. What are the implications of blocking PKRfunction and preventing the phosphorylation of eIF2a duringviral infection? Recent evidence suggests that inhibition ofPKR function is a key feature in the establishment of persistentviral infection. Lau and colleagues (489) examined EMCVinfection in established cell lines that were deficient in PKRexpression or in control cells that expressed normal levels ofPKR. EMCV normally mediates a cytolytic infection in vitrothat is characterized by massive apoptosis of permissive cells.However, EMCV infection of PKR-deficient cells conferredpersistent infection to this otherwise cytolytic virus. Althoughviral RNA translation was not directly examined in these stud-ies, the results suggest that constitutive disruption of PKR-dependent eIF2a phosphorylation may facilitate the establish-ment of persistent viral infection. Such a relationship betweenviral control of PKR and establishment of persistent infectionmay prove important for HCV infection. HCV mediates per-sistent infection within a majority of infected individuals (6).Persistent HCV infection is strongly associated with the devel-opment of hepatocellular carcinoma and lymphoproliferativedisorders (355, 356, 463). Recent observations now indicatethat viral persistence and disruption of host apoptosis arelinked to the block in eIF2a phosphorylation mediated by theHCV NS5A protein (134). Expression of NS5A in mammaliancells induces a block in eIF2a phosphorylation and concomi-tant stimulation of mRNA translation through NS5A-medi-ated repression of PKR. NS5A may contribute to HCV per-sistence by removing the translational blockade imposed byPKR-dependent eIF2a phosphorylation (Fig. 17). Consequent-ly, however, constitutive expression of NS5A rendered cells

FIG. 17. Translational control by HCV. (A) Structural representation of the HCV polyprotein. The individual positions of the polyprotein cleavage products areshown. NS5A (black region) from IFN-resistant HCV can bind and repress PKR (129, 133). (B) NS5A blocks PKR-dependent eIF2a phosphorylation. eIF2aphosphorylation from control (Neo) NIH 3T3 cell lines and those stably expressing NS5A from IFN-resistant HCV (NS5A-1A) or a nonfunctional NS5A mutant(DISDR) was assessed by single-dimension isoelectric focusing of cell extracts and anti-eIF2a immunoblot analysis (133). Cells were mock infected (lanes 1, 3, and 5)or infected with VSV (lanes 2, 4, and 6). Arrows denote the positions hypo- and hyper-phosphorylated isoforms of eIF2a (eIF2a and eIF2aP, respectively). Hyper-phosphorylated eIF2a is phosphorylated on serine 51 by PKR and is sufficient to block mRNA translation (61, 89).



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refractory to PKR-dependent apoptosis (134). These resultssupport previous studies indicating that eIF2a phosphorylationis an important component of cellular apoptotic signaling(436). The mechanisms by which eIF2a phosphorylation pro-mote apoptosis are not well understood. Current thinking pro-poses that the phosphorylation of eIF2a is required to blockthe synthesis of antiapoptotic gene products during the apo-ptotic response (450). More recently, however, high-level PKRexpression and PKR-dependent eIF2a phosphorylation havebeen associated with increased synthesis of proapoptotic effec-tor proteins, including Bax and Fas (16, 95). Thus, eIF2aphosphorylation may result in the selective translation of spe-cific proapoptotic mRNAs. Constitutive disruption of eIF2aphosphorylation that may occur during persistent HCV infec-tion may therefore render the host cell refractory to apoptoticsignaling. It is problematic for the host that disruption of eIF2aphosphorylation and the ensuing block in apoptotic signaling isassociated with oncogenic transformation (16, 134).

Cell growth control, eIF2a phosphorylation, and oncogenictransformation. Results from recent studies suggest that eIF2aphosphorylation may regulate cell growth, in part by enforcinga translational control and apoptosis checkpoint on cell pro-liferation. By this model, oncogenic potential is conferred topersistent viral infections in which the eIF2a checkpoint istargeted and constitutively disrupted. Thus, it is interestingthat a wide range of tumorigenic viruses possess mechanismsto repress PKR-dependent eIF2a phosphorylation during in-fection (reviewed in reference 131). Recent work suggests thatPKR activity and eIF2a phosphorylation are strictly regulatedduring the cell division cycle (495; M. Gale, Jr., C. Zhou,E. J. Firpo, M. G. Katze, A. Rudendsky, and B. R. Franza, Jr.,submitted for publication) and that inhibition of PKR functionresults in perturbation of cell cycle control (16, 95). Our studiessuggest that the NS5A protein from IFN-resistant HCV mayconfer an oncogenic potential to infected cells through theconstitutive inhibition of PKR (134). It is fitting to speculatethat inhibition of PKR function and disruption of eIF2a phos-phorylation may contribute to the development of hepatocel-lular carcinoma in patients persistently infected with HCV.

mRNA TRANSLATION AS A TARGET FORANTIVIRAL THERAPY

As our understanding of viral mRNA translation increases,it is becoming quite clear that viruses often utilize rather un-orthodox tactics to ensure their efficient mRNA translation.Such viral strategies that deviate from the processes of con-ventional cellular mRNA translation may represent potentialtargets for the therapeutic intervention in viral replication. Theutility of targeting translation for the development of anti-microbial therapeutics has been successfully demonstratedthrough the use of antibiotics such as tetracycline and eryth-romycin, which target bacterium-specific elements of transla-tion. The challenges to developing successful antiviral thera-peutics that target viral translational programming remain in(i) identifying translational targets that are specific to the virusand (ii) ensuring that host translation remains selectively un-disturbed by the therapeutic intervention. As is evident fromthe numerous examples cited in this review, viral translationalprogramming intersects with nearly every aspect of cellularmRNA translation, thereby making these challenges all themore spectacular. However daunting a task, therapeutic inter-vention in viral mRNA translation remains a promising arenafor the development of effective antiviral compounds, which inlarge part have been limited to viral polymerase or proteaseinhibitors.

Recent work has used antisense oligonucleotide strategies,dsRNA selection and targeting strategies, and direct mutagen-esis of viral mRNA to demonstrate that effective viral transla-tional targeting can be achieved. Here we briefly present someinteresting highlights in this developing field. This section fea-tures strategies that are under development as potential ther-apeutics to combat the global HCV pandemic. The develop-ment of antiviral drugs for HCV infection is problematic, dueto the high mutation rate of HCV and the enormous level ofquasispeciation that occurs during infection (46, 92). Thus, onecan expect that potential HCV therapeutics that target theviral polymerase, helicase, or protease activities may ultimatelybe of limited value, since drug-resistant strains will certainlyemerge. Indeed, this has already been demonstrated for HIV,which exhibits a similarly high mutation rate during infection(92). The roles of the HCV 59 and 39 UTRs in viral mRNAtranslation and replication and the fact that these regions arehighly conserved among the different HCV genotypes makethem viable targets for the development of antiviral therapies.Thus, strategies which target the function of the HCV IRESand the translation-stimulatory activity of the viral 39 UTR mayrepresent efficient means of blocking HCV protein synthesis.Moreover, the development of compounds that may disruptthe ability of the NS5A protein to bind and repress PKR mayserve to increase the efficacy of IFN and the existing IFNtherapeutic regimens. For further information on therapeutictargeting of viral mRNA translation, the reader is referred toa review by Harford, and references therein (182).

IRES-mediated translation is not a common feature amongcellular mRNAs, suggesting that it may represent a viabletarget for therapeutic intervention in viral mRNA translation.In support of this idea, Dasgupta and colleagues have identi-fied a cellular RNA that possess an IRES-inhibitory function(467). This 60-nt RNA, termed IRNA, was isolated fromS. cerevisiae and was first identified by examining the efficiencyof poliovirus IRES-mediated translation in S. cerevisiae. Theseinvestigators found that poliovirus IRES translation was blockedin yeast and that this was due to a trans-acting factor that couldsimilarly prevent poliovirus IRES translation when added totranslationally competent HeLa extracts (79). Purified IRNAwas shown to specifically inhibit IRES-mediated translationwithout having any effect on cap-dependent translation of cel-lular mRNAs. Interestingly, the IRNA was found to bind thecellular La protein (80), a major IRES-binding protein andeffector of IRES-mediated translation (23). Evidence for otherIRNA-polypeptide interactions was also demonstrated. Theseresults suggested that the IRNA functions as an IRES-bindingprotein competitor to block translation from the poliovirusIRES. Subsequent studies revealed that expression of theIRNA in hepatoma cell lines similarly rendered a block totranslation from the HCV IRES (81). Moreover, cells express-ing the IRNA were refractory to infection with chimeric po-liovirus under control of the HCV IRES. These results supportthe idea that the poliovirus and HCV IRES elements may binda similar repertoire of cellular proteins. Structure-functionanalysis of the IRNA indicates that specific secondary struc-tures are required for IRNA to bind cellular factors that pro-mote IRES function (467). Thus, it appears that the IRNAmay functionally mimic the IRES, at least in the context ofsecondary structure, and thereby compete for cellular proteinsthat mediate IRES translation. However, many questions re-main to be addressed regarding the nature and origin of theIRNA itself. What is the cellular function of this RNA species,and are there homologous sequences present in the cells ofhigher eukaryotes? How may this IRNA control or interferewith cellular gene expression? Until such questions are an-



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swered, the potential therapeutic value of IRNA sequenceswill be significantly limited.

The targeted disruption of HCV genome translation hassimilarly been achieved using antisense oligonucleotides. Eval-uation of the translation-inhibitory properties of a limited li-brary of chemically modified oligonucleotides, directed to var-ious regions of the HCV IRES and core protein-coding region,identified at least two antisense sequences that effectively in-hibited HCV gene expression (175). In these studies, the ex-pression of a truncated HCV genome in immortalized humanhepatocytes was ablated by hybridization of an antisense oli-gonucleotide corresponding to a region encompassing the ini-tiator AUG codon of the HCV core protein. Analysis of HCVRNA levels revealed that inhibition of genome expression wasachieved without reducing genomic RNA expression. These re-sults indicate that inhibition of HCV genome expression oc-curred at the level of translation and was not dependent on theactivation of endogenous RNase H activity by duplex RNA.The translational block imposed by antisense oligonucleotideshas yet to be confirmed by polyribosome analyses of HCVgenome expression in the presence or absence of oligonucle-otide treatment.

The possible utility of antisense oligonucleotides as an HCVantiviral therapy was demonstrated in a mouse model of HCVinfection (498). This system utilized a recombinant vacciniavirus expressing an HCV 59 UTR-core region construct fusedto the firefly luciferase gene. Translation of this HCV-core–luciferase construct was under control of the authentic HCVIRES. Mice infected with this vaccinia virus recombinant ex-hibited a block in liver-specific luciferase activity when treatedwith antisense oligonucleotides directed to the core proteininitiation codon and flanking sequences. Luciferase expressionremained high in infected mice that received oligonucleotidecontrols. These results are subject to the following criticisms:(i) they used a heterologous virus system, which may not faith-fully represent events of HCV infection, and (ii) the actualmechanism(s) contributing to inhibition of HCV (luciferase)expression was not determined. However, considering the enor-mous number of applications of antisense strategies and theefficiency with which antisense transcripts disrupt gene expres-sion (2, 262), antisense targeting of HCV replication remains aviable means of developing anti-HCV therapies. Taken togeth-er, these results indicate that antisense oligonucleotides mayprovide a potent mechanism by which to target viral mRNAtranslation as an antiviral therapy.

Another possible target of antisense-oligonucleotide strate-gies may be found within the HCV 39 UTR. As detailed above,this region of the HCV genome is highly conserved in all viralisolates, where it is thought to play an important a role ingenome replication. It is appropriate to speculate that anti-sense oligonucleotides directed to within the HCV 39 UTRmay disrupt transcription and block the translation-stimulatoryactivity induced by PTB binding (218). Finally, it should benoted that the use of antisense-oligonucleotide strategy to tar-get specific RNAs for degradation by the IFN-induced RNase,RNase L, has been achieved (317, 461). Although this strategyis not directly aimed at blocking viral mRNA translation, itpresents a viable option for targeting the HCV RNA forspecific degradation. In contrast to strategies that depend onRNA degradation by RNase H, a predominant nuclear pro-tein, RNase L is a resident cytoplasmic enzyme and would beavailable to disrupt HCV replication, which also takes place inthe cytoplasm (500). The possibilities for targeting the HCV 39UTR and the use of the RNase L pathway remain excitingareas of research into antisense oligonucleotides.

Similar to the use of antisense oligonucleotides, ribozymes,

which are enzymatic RNA molecules that catalyze the cleavageof RNA, can be constructed to target specific RNA sequences(145). Ribozymes have been shown to be effective in blockingtranslation directed from the HCV IRES, although this prob-ably occurs indirectly by RNA cleavage. Ribozymes con-structed to target conserved sites within the viral 59 UTRblocked the translation of a luciferase reporter protein un-der control of the HCV IRES and 59 UTR in a tissue culturesystem, with little or no apparent toxicity to the host cell (402).Ribozymes have the added advantage of being efficiently pack-aged and expressed by various viral vectors, including vacciniavirus, adeno-associated virus, and various retroviruses. More-over, ribozymes have been effective in catalyzing the degrada-tion of both positive and negative strands of the HCV RNA(478). Strand-specific targeting of HCV RNA by ribozymesmay thus hold promise for blocking the emergence of drug-resistant stains of HCV by eliminating new RNA variants.

Viral resistance to the current IFN-based therapeutic re-gimes for HCV infection is a major problem (121, 208, 263)and is in part responsible for the high frequency of persistentinfections within the HCV-infected population. As describedabove, HCV resistance to IFN has been attributed in part toviral repression of the IFN-induced protein kinase PKR. TheNS5A-PKR interaction may provide a useful therapeutic targetfor increasing the sensitivity to IFN in individuals who initiallyfail to respond to IFN therapy. Disruption of the NS5A-PKRinteraction may restore eIF2a phosphorylation and the trans-lational blockade imposed by PKR, thereby increasing theantiviral activity of IFN. Moreover, it is conceivable that res-toration of PKR-dependent eIF2a phosphorylation may proveuseful in reducing the incidence of cellular proliferative disor-ders associated with persistent HCV infection, since our resultsindicate that NS5A repression of PKR may provide an onco-genic potential to HCV (134).

CONCLUSIONS AND PERSPECTIVES

Investigations into the mechanisms and controls of viral pro-tein synthesis have led the way to understanding the processesof cellular mRNA translation. Analyses of viral systems haveintroduced us to a better understanding of cellular antiviralpathways and signaling processes that affect mRNA transla-tion. It is now clear that the translational control is an intimatepart of most, if not all, metabolic processes of the cell. Withthis said, it now becomes important to understand the mech-anisms of translational specificity; that is, how do cells andviruses impart translational control of specific mRNAs in a seaof translationally competent transcripts, and how do the extra-cellular environment and environmental cues effect mRNAtranslation?

Selective mRNA translation is a hallmark of many viral in-fections. While it is clear that viruses often encode mechanismsto disrupt cellular mRNA translation, the molecular mecha-nisms of selective viral mRNA translation under conditions ofhost protein synthesis shutoff remain poorly understood. Inparticular, the molecular mechanisms of IRES-mediated trans-lation have yet to be elucidated. What are the basal factors thatare required to support IRES-mediated translation? What isthe actual role of IRES-binding proteins, and how do theyfunction to stimulate viral mRNA translation? Similar ques-tions extend to the mechanisms of cap-dependent selectivetranslation. Identification of trans-acting factors that directlyinteract with viral RNA and understanding the implications ofsuch interactions for viral translational programming remainthe logical course of action. However, these approaches havebeen limited in scope by the nature of the traditional biochem-



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ical and molecular techniques often used when conductingsuch studies.

Recent advances in protein and nucleic acid analyses, suchas combined mass spectrometry and sequence database search-ing (170), tandem mass spectrometry (171), and the currentgenomic technologies (44, 56, 236, 299, 407), make these tech-niques viable tools for investigating the mechanisms of trans-lation control. The application of such techniques should allowfor characterizing novel RNA-protein interaction. Genomictechnologies, such as the use of high-density genome arrays,have already offered spectacular and sometimes surprising in-sights into the differential spectrum of cellular gene expressionunder various environmental conditions (88, 308, 350, 375, 408,434). Similar applications to assess the array of both viral andcellular gene expression in virus-infected cells are provingequally interesting. In particular, high-density genome arrayscan be used for the parallel identification of transcriptionallyand translationally regulated mRNAs (150, 502). Recent stud-ies have demonstrated the utility of this latter approach. Mor-ris and colleagues (504) separated cellular mRNAs from rest-ing or mitogenically activated fibroblast cultures into discretepools based on the number of mRNA-bound ribosomes. Byinterrogating cDNA microarray filters with probes generatedfrom the mRNA pools, these investigators found that transla-tional control of cellular mRNA, at least in the context ofmitogenic stimulation, was remarkably selective and represent-ed less than 1% of the mRNAs in this cross-sectional analysis.Taking this application a step further, Sarnow and colleagues(234) interrogated cDNA microarrays with probes derivedfrom polyribosome-associated mRNAs prepared from poliovi-rus-infected cells. This application allowed the investigators toidentify cellular mRNAs that could be translated independentof a functional eIF4F complex. Remarkably, it was found thatapproximately 2 to 3% of the mRNAs analyzed were associat-ed with polyribosomes under these conditions. When exam-ined for their ability to direct the cap-independent translationof a bicistronic reporter protein, it was shown that at least asubset of these mRNAs contained functional IRES sequenceswithin their 59 UTR. An emerging theme from these analysesis that mRNAs, whose gene products have been implicated ina variety of stress responses, are translated with little or no re-quirement for eIF4F. Thus, IRES-mediated translation may beprevalent among mRNAs that are involved in acute cellularresponses.

Development of an understanding of the acute cellular sig-naling pathways that modulate mRNA translation has nowattracted the attention of those in the signal transduction field(43). It has become very clear that cellular mRNA translationis controlled in response to specific environmental signals thatmodulate development, cell proliferation, and apoptosis. Re-cent evidence now indicates that viruses impinge on thesepathways during infection to promote viral replication. Animportant question now is how viral infection affects thesepathways and how this may contribute to viral pathogenesis.

Determination of the three-dimensional structure of trans-lation-regulatory proteins and translation factors representsanother fruitful area for future research. Structural determi-nation of translation factors is essential for a full understand-ing of the complexity and control of translation factor inter-actions and function. This was demonstrated by the recentelucidation of the crystal structure of eIF4E (318). As witheIF4E, translation factors play prominent roles in cell prolif-eration and malignancy (67, 267, 433). Understanding transla-tion factor structure may pave the way for rational drug designof anticancer and antiviral therapeutic compounds.

ACKNOWLEDGMENTS

We thank Marlene Wambach, Cecelia Boyer, and Dagma Daniel fortheir excellent administrative and technical support for the past severalyears. We thank the many members of the Katze laboratory, past andpresent, for their contributions to our work. We are grateful to indi-viduals who have collaborated with us over the years and who continueto share our interests in translational control. We thank Young WooPark for sharing major results prior to publication.

M.G. thanks the Helen Hay Whitney Foundation for outstandingpostdoctoral support. Work in the Gale laboratory is funded by the UTSouthwestern Endowed Scholars Program and by the Texas AppliedResearch Program. Work in the Katze laboratory is supported byNational Institutes of Health grants AI22646, RR00166, and AI41629;by Ribogene Corporation; and by the Gustave and Louise PfeifferResearch Foundation.

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