Immunology and Cell Biology (1996) 74, 523-526

Mechanisms of interference with the MHC class I-restrictedpathway of antigen presentation by herpesviruses

ANN B HILL

Department of Molecular Microbiology^ and Immunology. Oregon Health Sciences University, Portland,Oregon. USA

Summao' Herpesviruses are an ancient, ubiquitous family of DNA viruses, most of which share a lifestyle oflatently or persistently infecting a young host, and spreading to infect a new host a generation later. Mostherpesviruses interfere with antigen presentation via the MHC class I-restricted pathway of antigen presenta-tion, suggesting that impairment ofthe cytotoxic T lymphocyte response is necessary for the maintenance ofthis lifestyle. The diverse molecular mechanisms that have so far been discovered employed by Epstein-Barrvirus, herpes simplex viruses, and human and murine cytomegaloviruses are described in this review.

Key words: antigen presentation, cytomegalovirus, cytotoxic T lymphocyte, Epstein-Barr virus, herpessimplexvirus, herpesvirus, latency, MHC class I, natural killer cell, transporter associated with antigenprocessing.

The herpesviruses: what defines them as a family

Herpesviruses are a phylogenetically ancient family oflarge DNA viruses. The family recognized as herpesviri-dae includes the mammalian and avian viruses, whichshare a core set of recognizably homologous genes as wellas a conserved genome organization.' Even more ancientphylogenetically is the conserved herpesvirus virion struc-ture. Herpesviruses were divided initially on the basis ofcell tropism, and later on sequence comparison into threesubfamilies, alpha, beta and gamma.-̂ Alpha herpes-viruses are neurotropic; in humans these are herpes sim-plex viruses {HSV) types 1 and 2, and Varicella-zostervirus (VZV). The betaherpesviruses infect a multitude ofcell types in vivo; these are the cytomegaloviruses (CMV).More recently two newly discovered herpesviruses,human herpesvirus 6 (HHV-6) and HHV-7, have beenclassified as betaherpesviruses. The gammaherpesvirus ofhumans is the Epstein-Barr virus (EBV).

Herpesviruses also have much in common in terms oftheir lifestyle, that is, the mechanism by which they sur-vive and propagate within a species. Most herpesvirusesinfect their hosts in the first decade of life; the initialinfection usually causes mild or no symptoms. The virusestablishes latent or persistent infection ofthe host and, atleast for the well characterized viruses (HSV-1 and 2,CMV, VZV and EBV), the cellular site of latency or per-sistence is different to the primary cell in which efficientproductive replication occurs. Throughout the activehealthy life ofthe host new virus production occurs, not toan extent that it is noticed by the host but enough to

Correspondence; Ann B Hill, Department of Molecular Micro-biology and Immunology, L220, Oregon Heallh Sciences Univer-sity, 3181 SW Sam Jackson Park Rd. Portland, OR 97201, USA.

Received 3 September 1996; accepted 3 September 1996.

produce an inoculum capable of infecting a new individ-ual. Most infection of new hosts probably occurs from theadults in a child's immediate environment. Thus mostnew herpesvirus infection is from a host who was him/herself infected some decades earlier. This lifestyle hasbeen highly successful for the herpesviruses: most of thehuman herpesviruses affect most individuals of our spe-cies and. given the phylogenetic history of the herpes-viruses, the spread from generation to generation wouldappear to have occurred successfully for tens to hundredsof millions of years.

This extraordinary coexistence involves a close accom-modation with the host immune system. In order toachieve the generation to generation spread, herpesvi-ruses must avoid destruction by the immune system, andindeed must be able to replicate sufficiently to produce aninfective inoculum in the face of a fully primed immunesystem. Herpesviruses have evolved a multitude of mech-anisms to interfere with various aspects of the host im-mune response. Interference with the MHC classI-restricted pathway of antigen presentation is particularlycommon, and is the subject of this review.

However, the successful herpesvirus lifestyle is also ut-terly dependent on the normal functioning of the immunesystem. Primary herpesvirus infection in an immunodefi-cient infant is often fatal. When immunodeficiency occurslater in life, the inability to maintain control of latent/persistent virus makes herpesvirus infections, particularlyCMV, one ofthe most common causes of morbidity andmortality in the immuncompromized host. Thus theherpesvirus survival strategy, which depends on the in-fected host surviving to reproduce and pass the infectionon to a new generation, requires a healthy immune sys-tem. The dual nature of this interaction with the immunesystem needs to be borne in mind when attempting to

524 AB Htll

understand the complexity of mechanisms with whichherpesviruses interfere with immune function.

presumably constrains the effectiveness of viral interfer-ence manoeuvres which would abolish class I transport.

The class I antigen presentation pathway

The MHC class I-restricted antigen presentation pathwayis the immune system's way of monitoring the internalcontents of intact cells. MHC class I molecules bind pep-tides derived mostly from cytosolic and nuclear proteinsand transport them to the surface of cells where they canbe detected by the T cell receptor of CD8* T cells.^ CD8*T cells upon activation are capable of killing cells bearingthe MHC class I-peptide complex they recognize, andhence are commonly known as cytotoxic T lymphocytes(CTL). However, they also secrete a number of cytokinesand chemokines, and this function is also critical for theirantiviral effectiveness.

The processing of antigen for presentation by MHCclass I molecules begins in the cytosol. where protein isdegraded by the proteasome into short peptides. Peptidesare then transported into the endoplasmic reticulum (ER)by the transporter associated with antigen processing(TAP), The details of targeting of proteins for degradationand of the linkage between the proteasome and TAP arenot known. TAP transports preferentially peptides thatare of the right size and upwards for binding to class IMHC molecules, and which bear an appropriate C termi-nus for binding to the class I molecules of that species.Peptides delivered by TAP can bind to waiting nascentclass I molecules."*

Class I molecules consist of a polymorphic heavy chain,a light chain (beta-2 microglobulin) and the bound pep-tide. Class I heavy chain is a type I membrane glycopro-tein. co-translationally translocated into the ER where itacquires one or two N-linked glycans. After initial trim-ming of glycan residues, class I associates with the chap-eron calnexin. where it folds with beta-2 microglobulin(beta-2-m). On association with beta-2-m the complexachieves its final conformation (as detected by antibodies)but the association is unstable in the absence of peptidebinding. In human (but not mouse) class I may pass toanother chaperon at this point. The entire complex thenassociates with TAP. Again, several lines of evidence sug-gest that at least one other molecule is involved in theprocess at this point. Upon binding of a suitable peptide,heavy chain/beta-2-m and calnexin dissociate from TAP;the class I molecule then dissociates from calnexin and isable to leave the ER. Without peptide, most class I mole-cules remain in the ER. After leaving the ER, class Itravels through the Golgi, where its glycans are modified,and arrives at the cell surface, where it can interact withthe TCR and CD8 molecules of CTL. (The assembly ofclass I molecules has been recently reviewed in ref. 5.)

The class I-restricted antigen presentation pathwaythus serves to provide the immune system with a contin-uous survey of the protein content of the cytosol andnucleus. The immune system apparently has a backupsystem if the class I reporting fails. Some receptors of NKcells deliver an inhibitory signal upon binding to class Imolecules. The end result is that these NK cells will lysecells with inadequate levels of class I.'' This NK 'backup'

Molecular mechanisms of interference with class I inherpesvirus infections

Epstein-Barr virtis

EBV is a herpesvirus which enters a truly latent state, thatis, one in which no virus replication occurs. Several pat-tems of gene expression exist for EBV latency, but in all ofthem the gene EBNA-1 is expressed, and in one of themEBNA-1 is the only gene expressed. EBNA-1 is essentialfor maintenance of the viral episome during latency. It isalso the only one of the latency associated proteins againstwhich no cytotoxic T cells have ever been detected.EBNA-1 contains a long stretch of repeated glycine andalanine residues. When this gly-ala repeat stretch is re-moved, EBNA-1 specific responses can be elicited inmice. Furthermore, if the gly-ala repeat stretch is insertedinto another, normally antigenic protein. EBNA-4, therecombinant EBNA-4 is no longer able to be recognizedby CTL. Thus EBNA-1 is able to selectively prevent itsown processing for presentation by CTL while leaving thepresentation of other proteins intact. Cells latently in-fected with EBV expressing EBNA-1 are thus invisible tothe immune system.'

Herpes simplex virus

HSV types 1 and 2 encode a small cytosolic protein,ICP47, which binds to TAP and completely blocks itsability to transport peptides.^-^ ICP47 is encoded by animmediate early gene, US 12, and the blockade is effectivewithin 2 h of infection of human fibroblasts with HSV.*'°Blockade of TAP by ICP47 abolishes antigen recognitionby CTL. ICP47 is an 87 amino acid cytosolic protein. Itblocks TAP by binding to it with high affinity and a slowdissociation rate. In order to co-precipitate TAP withICP47, both subunits of the TAP heterodimer must bepresent. Both subunits of TAP are also required to trans-port peptides, and competition studies between ICP47and peptide for binding to TAP indicate that TAP andpeptides compete for a single binding site.'' Comparisonof sequences of ICP47 from HSV types 1 and 2 reveals ahigh degree of homology in the N-terminal two-thirds ofthe molecule, and almost no homology in the C terminalthird (B Manning & D McGeoch, unpubl. data, 1993). Asynthetic polypeptide corresponding to the N-terminal 53amino acids blocks TAP function as effectively as the fulllength molecule in a peptide transport assay using perme-abilized cells (B Galocha, A Hill & H Ploegh. unpubl.data, 1996).

In mouse models of HSV infection, HSV-specific CTLhave been easy to detect, a situation that contrasts withthe ditficuUy in detecting HSV-specific CTL in infectedhumans. ICP47 blocks antigen presentation to CTL inhuman but not mouse fibroblasts. Direct studies of TAPinhibition by ICP47 show that human TAP is blocked byICP47 with a half maximal efiiciencv of around 0.5 micro-

Herpesvirus interference with MHC elass I 525

Tnolar. In contrast, mouse TAP is not blocked by concen-trations of 10 micromolar.'' Studies of other species showthat old world primate TAP are blocked in a similarmanner to humans, but with increasing phylogenetic dis-tance the concentration of ICP47 causes 50% inhibitionincreases (P Jugovic & A Hill, unpubl. data, 1996).

HSV establishes latent infection in neurones, where noviral proteins are expressed. In its latent state the virus ishidden from the immune system both by virtue of theprivileged status of the neuron with regard to the immunesystem, and because of the paucity of antigen it produces.In contrast to EBNA-1 of EBV described above. ICP47acts to prevent CTL recognition not during latent infec-tion, but during the productive replication cycle of thevirus, which occurs in vivo in epithelial cells. We havesuggested that this may be necessary for the virus to rep-licate in sufficient numbers to infect a new host in the faceof a fully primed immune system.

Human cytomegalovirus

In human fibroblasts infected with human CMV(HCMV), class I heavy chain is synthesized at a normalrate but is degraded within minutes of synthesis.'-"'"* TwoHCMV gene products are independently capable of medi-ating this effect; they are encoded by the genes US2 andUS 11.' ^ In US 11 transfectants, class I is translocated intothe ER; it achieves its full length, is glycosylated, and asmall proportion becomes associated with beta-2-m. It isthen dislocated into the cytosol by an unknown mech-anism, where it is deglycosylated by an N-glycanase anddegraded by the proteasome.'^ US2, an apparently unre-lated gene, mediates the same effect.

US2 and USl 1 are early genes, that is, they depend onimmediate early encoded transcription factors for theirown transcription. Within the same genomic segment,Klaus Fruh et al. found two other genes encoding proteinswhich bind to class I MHC.'^ These are US3 and US6.US3 is an immediate early gene and, in US3 transfectants,class I MHC is retained in the ER. Thus in cells undergo-ing full lytic cycle infection with HCMV, class I is firstretained by US3, then degraded by US2 and USl 1.'^ TheUS6 gene product also binds to class I; its function is notknown.

Finally, in a different region of its genome, HCMVencodes a gene (UL18) which is homologous to class Iheavy chain.' ̂ It has been difficult to demonstrate a func-tion for this gene, or even to detect significant proteinproduct in HCMV-infected fibroblasts. However, theUL18 product associates with beta-2-m''^ and bindspeptides.-° Recent experiments have demonstrated thatin transfectants UL18 exerts a powerful inhibitory signalto human NK cell clones which recognise lack of self classI (HLA-C) (H Reyburn & J Strominger, unpubl. obs.,1996). It is thus likely that UL18 acts as a decoy toprevent NK-mediated destruction of HCMV-infectedcells which have lost class I expression due to the actionsof US2. 3 and 11. The NK decoy function of UL18remains to be demonstrated in the context of real infec-tion with HCMV.

The existence of this array of class I modulating genes

in HCMV provokes the question: why does the virus needsuch a complex arsenal? Questions on the role of each ofthese genes in the context of a real virus infection, inprimary infection, in latent/persistent infection and in thecell types involved therein, and in reactivated productiveinfection, are difficult to address in HCMV infection. Tounderstand the role of class I manipulation in the host-virus relationship I have turned in my own work to themurine cytomegalovirus model.

Murine cytomegalovirus

CMV are highly species-specific and are thought to haveco-evolved with their hosts. Thus in evolutionary timemurine CMV (MCMV) is as distant from HCMV as ismouse from human. Because of the much higher replica-tion rate of viruses compared to their hosts in this timethe genetic distance between the viruses is vastly greaterthan between their hosts; conserved features can be as-sumed to result from powerful selective pressure for theirconservation. It is therefore interesting to note that thenatural history of MCMV in terms of its host relationship,types of cells infected and pathogenesis is similar to thatof HCMV.

MCMV also interferes with antigen presentationthrough the class I pathway,•''"^^ although at present therewould seem to be little similarity in molecular mechan-isms with HCMV. An early gene of MCMV causes fullypeptide loaded class I molecules to be retained in the ER.This results in lack of presentation of epitopes within theimmediate early protein pp89, despite the continued syn-thesis of pp89. The mechanism by which MCMV retainsclass I in the ER is not known.

We have identified a second gene product in MCMV,encoded by the gene mO4. which binds to class I MHC(Kleijnen MF et al.. unpubl. data, 1996). M04 encodes a34kDa glycoprotein, most of which remains localized tothe ER. Gp34 binds to properly folded (beta-2-m associ-ated) class I in the ER, and travels with it to the cellsurface, where they remain tightly associated. The reten-tion of class I in the ER precedes the action ofgp34; laterin infection gp34 is synthesized in large quantities and lessclass I is retained in the ER. It remains to be testedwhether gp34 actually functions to rescue retained class Ifrom the ER. To date we have been unable to determine afunctional effect of gp34 in immune function. Neverthe-less it appears that, like HCMV, MCMV's intervention inthe class I pathway is multifaceted and finely regulated.The MCMV model offers the opportunity to determinethe effect of these manoeuvres on the host virus relation-ship.

Finally, as has been reported, like HCMV, MCMV alsoencodes a class I homologue.-^-^^ Again, the experimentalmanipulability of the MCMV model should enable thebiological function of this molecule to be readily deter-mined

Conclusion

Investigation of the interaction of herpesviruses with theclass I antigen presentation pathway has revealed an un-

526 AB Hill

expected diversity of mechanisms, and there are undoubt-edly many more to be discovered. It seems probable thatthese mechanisms will throw light on those parts of theclass I antigen presentation pathway which are not yetfully described. HCMV US2 and USll will almost cer-tainly reveal a normal cellular mechanism by which un-wanted proteins in the ER are degraded. In addition, it isto be hoped that elucidating these mechanisms will helpus to understand how herpesviruses maintain their life-style of persistence within a host and spread to new hostsacross the generations.

References

1 McGeoch DJ, Cook S, Dolan A, Jamieson FE, Telford EA.Molecular phylogeny and evolutionary timescale for thefamily of mammalian herpesviruses. / Molec. Bioi 1995;247: 443-58.

2 Roizman B. Herpesviridae: A brief introduction. In: FieldsBN, Knippe DM (eds) Virology, Vol. 2. New York: RavenPress Ltd 1990-. 1787-94.

3 Townsend A. Bodmer H. Antigen recognition by classI-restricted T lymphocytes. Annu. Rev. Immunoi 1989; 7:601-24.

4 Hecmels M-T, Ploegh HL. Generation, translocation andpresentation of MHC class I-restricted peptides. Annu. Rev.Biochem. 1995; 64: 463-91.

5 Williams DB, Vassilakos A, Suh W-K. Peptide presentationby MHC class I molecules. Trends Cell Bioi 1996; 6: 267-73.

6 Yokomaya WM. Natural killer cell receptors specific formajor histocompatibility complex class I molecules. Proc.Natl Acad. Sci. USA 1995; 92: 3081-5.

7 Levitskaya J. Coram M. Lcvitsky V el ai Inhibition of anti-gen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 1995; 375: 685-8.

8 York IA. Roop C. Andrews BW, Riddell SR, Graham FL,Johnson DC. A cytosolic herpes simplex virus proteininhibits antigen presentation to CD8* T lymphocytes. Cell\994:11: 525-35.

9 Hill AB. Jugovic P. York I et ai Herpes simplex virus turnsoff the TAP to evade host immunity. Nature 1995; 375:411-15.

10 Hill AB. Lee SP, Haurum JS et al. Class 1 MHC-restrictedcytotoxic t lymphocytes specific for EBV nuclear antigens failto lyse the EBV-transformed B-lymphoblastoid cell linesagain.st which they were raised. / Exp. Med. 1995; 181:2221-18.

11 Tomazin R, Hill AB, Jugovic P et al. Stable binding of theherpes simplex virus ICP47 protein to the peptide bindingsiteofTAP. EMBOJ. 1996; 15: 3256-66.

12 Beersma MFC. Bijlmakers MJE. Ploegh H. Human cytome-galovirus down-regulates HLA class I expression by reducingthe stability of class 1 heavy chains. / Immunoi 1993; 151:4455-64,

13 Warren AP, Ducroq DH, Lehner PJ, Borysiewicz LK. Hu-man cytomegalovirus-infected cells have unstable assemblyof major histocompatibitity complex class I complexes and

are resistant to lysis by cytotoxic T lymphocytes. J. Virol.1994; 68: 2822-9.

14 Yamashita Y, Shimokata S, Saga S, Mizuno S, Tsurumi T.Nishiyama Y. Rapid degradation of the heavy chain of classI major histocompatibility complex antigens in the endo-plasmic reticulum of human cytomegalovirus-infected cells./ Viroi 1994; 68: 7933-43.

15 Jones TR. Hanson LK. Sun L, Slater JS. Stenberg RM,Campbell AE. Multiple independent loci within the humancytomegalovirus unique short region down-regulate expres-sion of major histocompatibiiity complex class I heavychains./ Viroi 1995; 69: 4830-41.

16 Wiertz EJHJ, Jones TR, Sun L, Bogyo M, Geuze HJ, PloeghHL. The human cytomegalovirus USll gene product dis-locates MHC class 1 heavy chains from the endoplasmicreticulum to the cytosol. Cell 1996; 84: 769-79.

17 Ahn K. Angulo A. Ghazal P. Yang Y. Fruh K. Human cy-tomegalovirus inhibits antigen presentation by a sequentialmultistep process. Proc. Natl Acad. Sci. USA 1996; 93:10990-5.

18 Beck S. Barren BG. Human cytomegalovirus encodes aglycoprotein homologous to MHC class-I antigens. Nature1988; 331: 269-72.

19 Browne H, Smith G, Beck S, Minson T. A complex betweenthe MHC class I homologue encoded by human cytomegalo-virus and beta 2 microglobulin. Xature 1990; 347: 770-2.

20 Fahnestock ML, Johnson JL, Feldman RM, Neveu JM. LaneWS, Bjorkman PJ. The MHC class I homolog encoded byhuman cytomegalovirus binds endogenous peptides. Immu-nity 1995; 3: 583-90.

21 del Val M. Volkmer H. Rothbard JB et al. Molecular basisfor cytolytic T-lymphocyte recognition of the murine cyto-megalovirus immediate-early protein pp89. / Viroi 1988;62: 3965-72.

22 Campbell AE. Slater JS, Cavanaugh VJ, Stenberg RM. Anearly event m murine cytomegalovirus replication inhibitspresentation of eellular antigens to cytotoxic T lymphocytes.J. Virol. 1992; 66: 3011-17.

23 del Val M, Hengel H, Hacker H et ai Cytomegalovirusprevents antigen presentation by blocking the transport ofpcptide-loaded major histocompatibility complex class Imolecules into the medial-Golgi compartment. / Exp. Med.1992; 176; 729-38.

24 Thale R, Lucin P, Schneider K, Eggers M. Koszinowski UH.Identification and expression of a murine cytomegalovirusearly gene coding for an Fc receptor. J. I'irol. 1994; 68:7757-65.

25 Reddehase MJ, Eibi MR, Keil GM. Koszinowski UH. Late-phase expression of a murine cytomegalovirus immediate-early antigen recognized by cytolytic T lymphocytes. J. I 'iroi1986; 60: 1125-9.

26 Rawlinson WD, Farrell HE. Barrell BG. Analysis of thecomplete DNA sequence of murine cytomegalovirus. /I'iroi 1996 (in press).

27 Davis-Poynter NJ. Farrell HE. Masters of deception: A re-view of herpesvirus immune evasion strategies. Immunol.Cell Bioi 1996; 74: 513-21.