Communication Vol. 268, No. 26, Issue of September 15, pp. 19200-19203, 1993 THE JOURNAL OF BIOLOCI~AL CHEMISTRY

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Mapping the Cleavage Site in Protein Synthesis Initiation Factor eIF-4y of the 2A Proteases from Human Coxsackievirus and Rhinovirus*

(Received for publication, June 21, 1993, and in revised form, July 7, 1993)

Barry J. LamphearS, Riqiang YanSl, Fang Yang$, Debra Waters$, Hans-Dieter Liebid, Hannes Klumpll, Ernst Kuechlerll, Tim Skernll, and Robert E. RhoadsSII From the Wepartment of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreueport, Louisiana 71130-3932 and the Plnstitut fur Biochemie der medizinischen Fakultat der Universitat Wien, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria

The rate-limiting step of eukaryotic protein synthesis is the binding of mRNA to the 40 S ribosomal subunit, a step which is catalyzed by initiation factors of the eIF-4 (eukaryotic initiation factor 4) group: eIF-4A, eIF-4B, eIF-4E, and eIF-4y. Infection of cells with picornaviruses of the rhino- and enterovirus groups causes a shut-off in translation of cellular mRNAs but permits viral RNA translation to proceed. This change in translational specificity is thought to be mediated by proteolytic cleavage of eIF-4y, which is catalyzed, directly or indi- rectly, by the picornaviral2A protease. In this report we have used highly purified recombinant 2A protease from either human Coxsackievirus serotype B4 or rhinovirus serotype 2 to cleave eIF-4y in vitro in the eIF-4 complex purified from rabbit reticulocytes. Neither the rate of cleavage nor fragment sizes were affected by addition of eIF-3. The NH,- and COOH-terminal fragments of eIF4y were separated by reverse phase HPLC and identified with specific antibodies, and the NH,-terminal sequence of the COOH-terminal fragment was determined by au- tomated Edman degradation. The cleavage site for both proteases is 479GRPALSSR 1 GPPRGGPG4” in rabbit eIF-47, corresponding to 478GRTTLSTR 1 GPPRGGPG493 in human eIF-47.

The translation of all cellular and most viral mRNAs in eu- karyotic cells requires initiation factors of the eIF-4l group

* This work was supported by Research Grant GM20818 from the National Institute of General Medical Sciences, National Institutes of Health (to R. E. R,), a grant from the Austrian Heart Foundation (to T. S.), and grants from the Austrian Science Foundation and Boehringer Ingelheim (to E. K.). The costs of publication of this article were de- frayed in part by the payment of page charges. This article must there- fore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession number(s) L22090. The nucleotide sequence(s) reported in this paper has been submitted

University, New York, NY 10021. 5 Current address: Laboratory of Molecular Cell Biology, Rockefeller

and Molecular Biology, Louisiana State University Medical Center, I/ To whom correspondence should be addressed Dept. of Biochemistry

1501 Kings Hwy., Shreveport, LA 71130-3932. The abbreviations used are: eIF, eukaryotic initiation factor; CVB4,

(reviewed in Refs. 1-31. These polypeptides collectively recog- nize the m7GTP-containing cap, unwind secondary structure at the expense of ATP, and facilitate binding of the 40 S ribosomal subunit, although the order in which these events occur is not known (reviewed in Ref. 4). The eIF-4 polypeptides consist of eIF-4A (a 46-kDa RNA helicase), eIF-4B (a 67-kDa RNA-bind- ing protein), eIF-4E (a 25-kDa cap-binding protein), and eIF-4y (a 154-kDa protein of unknown function). Several different complexes of eIF-4 polypeptides have been isolated, the best studied being eIF-4 (also known as eIF-4F), consisting of eIF- 4E, eIF-4A, and eIF-4y (5, 6).

The overall rate of translation is profoundly affected by both the intracellular levels and phosphorylation states of eIF-4 polypeptides (reviewed in Ref. 7). One of the most dramatic physiological changes in translation rate occurs upon picor- naviral infection (8, 9). During their replication, many mem- bers of the picornaviridae (all genera except the cardioviruses) modify the ability of the host cell to translate capped mRNAs (10). This process is proposed to be mediated by the virally induced cleavage of eIF-4y, although this hypothesis has been challenged (11, 12). Another point of contention is whether eIF-4y is cleaved by a quiescent cellular protease that is acti- vated by the 2A protease of entero- and rhinoviruses (13-15) or is cleaved by 2A protease itself (16).

Definitive evidence that eIF-4y cleavage is responsible for host cell shut-off and resolution of the question of direct versus indirect proteolytic cleavage will require molecular genetic studies with modified forms of eIF-47. To this end, we have determined the site of cleavage in eIF-4y produced by in vitro incubation with 2A protease from human Coxsackievirus sero- type B4 (CW34) and human rhinovirus serotype 2 (HRV2).

EXPERIMENTAL PROCEDURES Materials-Rabbit reticulocyte lysate was prepared as described (17).

m7GTP was purchased from Sigma. The C4 column for reverse phase separation of eIF-4y cleavage products was obtained from Vydac (Hes- peria, CA). m’GTP-Sepharose was purchased from Pharmacia LKB Biotechnology Inc. Horseradish peroxidase-conjugated goat anti-rabbit IgG was obtained from Vector Laboratories (Burlingame, CA). All other chemicals were of reagent grade.

Purification of Proteins-Preparation of ribosomal salt wash from rabbit reticulocyte lysate and purification of eIF-3 and eIF-4 from ribo- somal salt wash were as described (19). Expression and purification of recombinant HRV2 and CVB4 2A protease were as described (16).

Cleavage of elF-4yCleavage reactions for CVB4 2A protease con- tained protease and eIF-4 at the concentrations indicated in the figure legends, 20 mM MOPS, pH 7.6, 10% glycerol (v/v), 200 mM KC1,0.25 mM dithiothreitol, 0.1 mM EDTA, 0.05% Tween 20, and, where indicated, eIF-3. Cleavage reactions for HRV2 2A protease contained the protease (40 pdml), eIF-4 (115 pdml), 50 mM Tris-HC1, pH 8.0, 5% glycerol (v/v), 100 mM NaC1,5 mM dithiothreitol, and 1 mM EDTA. Incubations were at 30 “C for either 10 min (HRV2) or the times indicated in the figure legends (CVB4).

Separation of eZF-4y Cleavage Products and Amino-terminal Se- quence Analysis-Cleavage products resulting from incubation of eIF-4 with 2A protease were fractionated by reverse phase HPLC on a Waters model 625 LC System. Reactions containing eIF-4 cleaved by CVB4 2A protease (2 ml) or HRVB 2A protease (0.3 ml) were applied directly to a 0.45 x 15-cm C4 column equilibrated in Buffer A (0.1% aqueous triflu- oroacetic acid). The column was developed with 5 ml of Buffer A, a 45-ml

human Coxsackievirus serotype B4; HPLC, high pressure liquid chro- matography; HRVB, human rhinovirus serotype 2; MOPS, 3-(N-mor- pho1ino)propanesulfonic acid; PAGE, polyacrylamide gel electrophore- sis; cp, cleavage product.

19200

2A Protease Cleavage Site of eZF-4y 19201

linear gradient of Buffer A to 80% Buffer B (0.1% trifluoroacetic acid in 95% acetonitrile), 2 ml of 80% Buffer B, a 2-ml gradient to 100% Buffer B, 2 ml of 100% Buffer B, and a 2-ml gradient back to 0% Buffer B. Cleavage products derived from the COOH terminus were subjected to automated Edman degradation using an Applied Biosystems model 470A sequenator.

Electrophoresis and Western Blotting-SDS-PAGE was performed us- ing the Laemmli buffer system (20) for 1.5 h at 100 V; protein bands were visualized after either staining with silver (21) or Western blot- ting. For the latter analysis, unstained gels were transferred to poly-vi- nylidene difluoride membranes using a Bio-Rad Mini Trans-Blot cell and electrode buffer (20) as per manufacturer's recommendations. Anti- peptide 6 antibody was generated against a COOH-terminal eIF-4y peptide and anti-peptide 7, against an NH,-terminal peptide as de- scribed previously (18). Detection of immunoreactive species was per- formed as described previously (18) with development enhanced by inclusion of CoCI2 (22).

RESULTS AND DISCUSSION Zn Vitro Cleavage of eZF-&Addition of recombinant 2A pro-

tease from CVB4 and HRV2 to highly purified eIF-4 resulted in the appearance of a series of faster migrating bands immuno- logically related to eIF4y. presumed to be proteolytic frag- ments (16). The same study described several experiments in- dicating that eIF4y is cleaved directly by 2A protease, rather than through activation of an endogenous cellular protease.

To analyze further this proteolytic reaction, eIF-4 purified from rabbit reticulocytes was incubated with recombinant CVB4 2A protease and the reaction time course analyzed (Fig. 1). Commencement of proteolysis was apparent after 5 min as judged by the disappearance of the 200-220-kDa bands of eIF-4y and appearance of the characteristic cleavage products ranging in size from 100 to 130 kDa (cp, and cpb) (18, 24-26). Four major cleavage products were clearly detected in the sil- ver-stained gel shown (Fig. lA). eIF-4 incubated for 60 min in

A. - dF-3 + clF-3 - + # 0 5 IO 20 40 60 N 0 5 10 20 40 60 N 16hr

4 -

FIO. 1. Time c o r n of eIF4y cleavage by CVB4 2.4 protenee. Reactions contained eIF-4 (25 pg/ml) and recombinant CVB4 2A prote- ase (10 pg/ml) and were performed as described under 'Experimental Procedures" except that the KC1 concentration was 160 mM. Where indicated (- and +), eIF-3 (58 pg/ml) was also included. Aliquota (5 pl) were removed at the times indicated (in min) and subjected to SDS- PAGE on an 6.5% gel followed by either staining with silver nitrate (panel A ) or immunoblotting with anti-peptide 6 (la), an antibody against a synthetic peptide derived from the COOH terminus of eIF-4y (panel B). Lanes N contain eIFdy incubated for 60 min in the absence of 2A protease under the reaction conditions described above. Molecular masses based on standard proteins are indicated at left. cp. and cpb indicate the position of NH,-terminal and COOH-terminal fragments of eIF-ly, respectively.

the absence of protease was unaltered (lane N), showing that the proteolytic activity was not present in the eIF-4 prepara- tion. Comparable results were obtained using HRV2 2A prote- ase (data not shown). The pattern was not changed by over- night incubation (Fig. lA, 16 hr).

Wyckoff et al. (27) have presented evidence that eIF-3 is required for the cleavage of e IF4y by poliovirus 2A protease. This initiation factor of 550 kDa contains seven primary polypeptides and several secondary polypeptides derived by proteolysis (1). To determine whether eIF-3 affected either the rate or site of cleavage, we repeated the time course in the presence of this factor at a concentration equimolar to that of eIF-4 (Fig. lA, +eZF-3 ). The rate of appearance and mobilities of eIF-4y cleavage products were unchanged. Additionally, no cleavage of eIF-4A, eIF-4E, or the various polypeptides of eIF-3 was observed (Fig. lA and data not shown). Similar results were obtained when the molar ratio of eIF-3 to eIF-4 was in- creased to 13 (data not shown). The reason for this apparent discrepancy with the study of Wyckoff et al. (27) is not known, but it may reflect a difference in the purity of components used in the two studies or a difference between poliovirus and Cox- sackievirus 2A proteases.

Despite the complexity of products, we propose that the 2A proteases have only a single cleavage site in eIF-4y. The mi- gration of eIF-4y on SDS-PAGE is heterogeneous, with three major, closely spaced forms apparent (19.24,29), but the cause of this is unknown. Cleavage by poliovirus in vivo produces three major fragments detected with an antibody directed against NH2-terminal peptide sequences (collectively referred to as cp.) but only a single fragment (Q,) detected with an antibody against COOH-terminal sequences (18). These results suggest that the heterogeneity in the intact eIF-4y polypeptide is localized to the NH2-terminal segment. To determine if Cox- sackievirus 2Aprotease behaved similarly in vitro, we analyzed the polypeptides in Fig. lA with the COOH-terminal antibody (Fig. 1B). A single COOH-terminal product was detected, a p pearing simultaneously with the NH2-terminal products. The kinetics of appearance of Q, were not affected by eIF-3, and no further cleavage occurred even after overnight incubation (Fig. 1 B , 16 hr). Interestingly. we have consistently observed with purified rabbit e IF4y preparations that the antibody reacted much more strongly with cpb than with intact eIF4y, suggest- ing that the epitope may be obscured in the intact protein. As a further argument that 2A protease has a single cleavage site, all cleavage products appeared simultaneously (Fig. LA), and the pattern did not change in complexity during incubation, as would be expected if there were multiple cleavages and inter- mediate products.

I t has been shown previously that cleavage products of eIF- 4y, produced by poliovirus infection of HeLa cells, co-migrate electrophoretically with products formed aRer infection of HeLa cells by either human rhinovirus (28) or Coxeackievirus (10). In addition, in vitro cleavage of e IF4y by extracts mn- taining poliovirus 2A protease yields products that co-migrate with cleavage products generated in vivo during poliovirus in- fection (10, 27). These results support the view that all three viral proteases cleave at the same site and also that in vitro cleavage of eIF-4y reflects the in vivo situation. The ability to cleave eIF-4y in vitro in a system containing only purified com- ponents enabled us to define the cleavage products biochemi- cally.

Purifiatwn and Zdentifiatwn of the Carboxyl-terminal Fragment of eZF-4yPurified eIF-4 was incubated with either CVB4 or HRV2 2A protease under the reaction conditions de- scribed under 'Experimental Procedures." The reaction was terminated when less than -2% of the intact eIF4y remained. Reaction products were resolved by reverse phase HPLC on a

19202 2A Protease Cleavage Site of eIF-4y

FIG. 2. Separation of eIF4y cleav- age products by reverse phase HPLC. eIF-4 (40 pg/ml) cleaved in a total reaction volume of 2 ml with CVB4 2A protease (60 pg/ml) was loaded directly on a C4 col- umn. Panel A shows SDS-PAGE analysis on 6.5% gels followed by silver staining of fractions obtained. U, C, and FT repre- sent lanes containing unreacted eIF-4, CVB4-cleaved eIF-4, and flow-through (pooled fractions 6-8), respectively. Panel B represents the eluting proteins as mon- itored by absorbance a t 280 nm. Peaks not identified by SDS-PAGE in panel A were identified in separate runs as buffer com- ponents (fractions 6-8, 14, 17, 18, 25-28, 41, 46, and 55), 2A protease (fraction 291, and eIF-4E (fraction 39).

A. Silver Stain B. N-term Ab

kDa U C 30 43 U C 30 43

A. kDa

200

97

88

43

u c [ -

Fr'25282728293031 32333435383730394041 4243444546

8.

F 0 m

0 N

0

C. C-term Ab

u c 3043

I I

Fro. 3. Identification of HPLC fractione by immunoblotting. Selected HPLC fractions (see Fig. 2) were subjected to electrophoresis on 6.5% gels. Gels were stained with silver (panel A ) or transferred to polyvinylidene difluoride membrane and probed with anti-NH,-termi- nal (panel B ) or anti-COOH-terminal (panel C ) anti-peptide antibod- ies. Lanes U and C contain unreacted and CVB4 2A protease-cleaved eIF-4, respectively. Lanes 30 and 43 contain aliquota from HPLC frac- tions 30 and 43, respectively.

C4 column, subjected to SDS-PAGE, and detected by silver staining. The results obtained with CVB4 2A protease are shown in Fig. 2; similar results were obtained with HRV2 2A protease (data not shown). The three slowest migrating frag- ments of eIF-4y eluted together in fraction 30, whereas the fastest migrating fragment eluted in fractions 42-44. Interest- ingly, although the latter fragment had the weakest silver- staining characteristics of the eIF-4y fragments, it possessed the greatest absorbance at 280 nm. eIF-4Aeluted in fraction 40. As the polyacrylamide concentration of 6.5% was employed to resolve the various fragments of eIF4y, polypeptides below 30 kDa were not resolved. Identification of the other peaks in the chromatogram was made from additional HPLC runs with in- dividual components of the reaction mixture (see figure leg- end).

HPLC fractions containing eIF-4y cleavage producta were tested with antibodies raised against synthetic peptides repre- senting NH2- and COOH-terminal sequences of eIF-4y (18). Anti-NH2- and COOH-terminal antibodies recognized different subsets of fragments (Fig. 3). The fragments co-eluting in frac-

1 1 1 1 1 1 1 1 1 1 1 1

10 20 30 40 50 80 Fraction Number

TABLE I In vitro cleavage site of CVB4 2.4 protease and HRV2 2A protease in

rabbit eIF-4y

Source of sequence Sequence

Rabbit eIF-4y cpb;

Rabbit eIF-4y c h ; XPPRGGPGXELPRG"

Rabbit e IF4y cDNA" "'GRPALSSR 1 GPPRGGPGGELPRG"'

HRV2 polyprotein' '"TRPIITTA 1 GPSDMYVHH"R CVB4 polyproteind "'ERASLITT 1 GPYGHQSG"'

CVB4 2A digestion

HRV2 2A digestion

GPPRGGPG

Human eIF-4y CDNAb "'GRTTLSTR 1 GPPRGGPGGELARG4'9

See footnote 2. Ref. 18.

e Ref. 31. Ref. 32.

'X indicates that an unambiguous assignment could not be made.

tion 30 were all recognized by the anti-NH2-terminal antibody and not by the anti-COOH-terminal antibody, indicating they were derived from the N H 2 terminus. Conversely, the fragment eluting at fraction 43 was specifically recognized by the anti- COOH-terminal antibody and was not by the anti-NH2-termi- nal antibody. Fragments generated by H W 2 2A protease gave identical results (data not shown). Therefore, reverse phase HPLC under these conditions readily resolves cp. and cpb. A minor product of -80 kDa, which was also recognized by anti- bodies against the COOH terminus, appeared only after re- verse phase HPLC (Fig. 3C, lane 431, but was not apparent in the original digest (Fig. 1 B ) . It is possible that it resulted from a cleavage by CVB4 2A protease at a second site. Alternatively, as i t was detectable only after HPLC. it may have resulted from breakdown of cpb under the acidic conditions used (pH 2).

Amino-terminal Sequence Analysis-HPLC fractions con- taining cpb generated upon cleavage by CVB4 or HRV2 2A protease were subjected to automated Edman degradation for determination of NH2-terminal sequence (Table I). The results indicate that cleavage ( 1 ) of rabbit eIF-4y by both proteases

2A Protease Cleavage Site of eIF-4y 19203

occurs at 479GRPALSSR 1 GPPRGGPG494. As there is consid- erable similarity in the amino acid sequences of rabbit2 and human eIF-4y (18), it is likely that the site determined for the rabbit protein is valid for human eIF4y and corresponds to the sequence 478GRTTLSTR J GPPRGGPG493. From the amino acid sequence of eIF4y and the cleavage site determined above, it can be calculated that cpb would be 102 kDa, consist- ent with the mobility of the -100-kDa cpb fragment on SDS- PAGE. Furthermore, this indicates that the portion of the eIF-4y molecule that causes the intact 154-kDa protein to mi- grate aberrantly is located somewhere in the NH,-terminal one-third.

Relationship of the eIF-4y Cleavage Site to Substrate Deter- minants for HRV2 2A Protease-Several studies have sug- gested that 2A protease cleaves eIF-4y in vivo by activation of an endogenous protease (13-15). However, Liebig et al. (16) have argued for a direct cleavage. An essential precondition for direct cleavage is the ability of 2A protease to recognize the cleavage site assigned on rabbit and human eIF-47. Knowledge of the cleavage site now permitted comparison with previously determined substrate requirements (23, 30).

For HRVB 2A protease, essential residues in substrate rec- ognition have been determined by systematic replacement of amino acids in the viral polyprotein (30) or in synthetic pep- tides (23). In both cases, the occupancy of P2 and P1’ was critical for cleavage; at P2, of 16 substituted peptides tested, only Ser, Asn, or Arg could replace the wild type Thr. The P1’ residue was even more restrictive, with only the wild type Gly being accepted (23h3 In contrast, several amino acids could be tolerated at the P1 site (23, 30).

Table I shows that the 2A protease cleavage sites on rabbit and human eIF-By satisfy these requirements, both containing Gly at Pl’, Ser or Thr at P2, and the well tolerated Arg residue at P1. Moreover, similarity is evident with the cleavage sites of both proteases at P7 (Arg) and P2’ (Pro), positions that are also important in determining cleavage (23).3 Further similarity exists between both eIF-4y cleavage sites and the cleavage site of CVB4 polyprotein at P4 (Leu) and between the rabbit eIF-4y site and the HRVB cleavage site at P6 (Pro; see Table I). Inter- estingly, although the P’ regions of human and rabbit eIF-4y are identical from P1’ to Pll’, there is little similarity with the viral cleavage sites. This is consistent with the fact that P’ residues play only a minor role in influencing cleavage effi- ciency on peptide substrates (23).

Knowledge of the cleavage site of e IF4y shown in Table I resolves the difficulty raised by the poor cleavage of HRV2 2A protease on peptides derived from the cleavage sites of other rhino- and enterovirus serotypes (23, 30). The inference of these studies was that a common sequence on the cellular tar- get of 2A proteases could not be defined. Given that most 2A cleavage sites have a negatively charged residue at P7 or P6; Thr, Asn, or Ser at P2; and Gly at Pl’ , it is probable that most 2Aproteases will cleave eIF-4y at the position determined here.

W. Sommergruber, H. Ahorn, A. Zoephel, F. Fessl, J. Seipelt, D. R. Yan, W. He, and R. E. Rhoads, manuscript in preparation.

Blaas, E. Kuechler, H. Klump, H.-D. Liebig, and T. Skern, manuscript in preparation.

Furthermore, the unusual nature of the P’ region (containing 5 Gly and 3 Pro residues in a space of nine residues) may induce a conformation suitable for 2A protease cleavage.

Our finding that the y subunit of eIF-4 purified from rabbit reticulocytes could be cleaved in vitro by 2A protease provides additional support for the direct cleavage of eIF-4y in vivo. However, the possibility could be raised that a second protease activity is present as a contaminant in the eIF-4, or is an inherent activity of eIF-4 itself. In experiments to be published e l~ewhere ,~ a 16-mer peptide matching the human eIF-4y cleavage sequence was synthesized and shown to be cleaved by both viral proteases. This argues against participation of an endogenous cellular protease, since the proteases were from bacterial sources and the peptide was chemically synthesized.

Acknowledgments-We thank Dr. Carol Beach (University of Ken- tucky) for performing sequence analysis of eIF-4y cleavage products and Dragana Jugovic for expert technical assistance.

REFERENCES

2. Thach, R. E. (1992) Cell 88, 177-180 1. Hershey, J. W. B. (1991)Annu. Reu. Biochem. 60,717-755

3. Merrick, W. C. (1992) Microbiol. Reu. 68,291415 4. Rhoads, R. E. (1991) in Dunslation in Eukaryotes (Trachsel, H., ed) pp. 109-

5. Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. 148, CRC Press, Inc., Boca Raton, FL

(1983) J. Biol. Chem. 268,5804-5810 6. Edery, I., Humbelin, M., Darveau, A,, Lee, K. A. W., Milburn, S., Hershey, J. W.

7. Rhoads, R. E. (1993) J. B i d . Chem. 288,3017-3020 B., Trachsel, H., and Sonenberg, N (1983) J. Biol. Chem. 258,11398-11403

8. Sonenberg, N. (1990) Curr. lbp. Microbiol. Zmmunol. 181, 23-47 9. Rueckert, R. R. (1990) in Virology (Fields, B. N., and Kneipe, D. M., eds) 2nd

10. Lloyd, R. E., Grubman, E. M. J., and Ehrenfeld, E. (1988) J. Virol. 82, 4 2 1 6 Ed., Vola. 1 and 2, pp. 507-548, Raven Press, New York

4223 11. Bonneau, A,”., and Sonenberg (1987) J. Virol. 61,986990 12. Perez, L., and Carasco, L. (1992) Virology 189, 17S186 13. Lloyd, R. E., lbyoda, H., Etchison, D., Wimmer, E., and Ehrenfeld, E. (1986)

14. Kriiusslich, H.-G., Nicklin, M. J. H., lbyoda, H., Etchison, D., and Wimmer, E.

15. Wyckoff, E. E., Lloyd, R. E., and Ehrenfeld, E. (1992) J. Virol. 68,2943-2951 16. Liebig, H.-D., Ziegler, E., Yan, R., Hartmuth, K., Klump, H., Kowalski, H.,

Blaas, D., Sommergruber, W., Frasel, L., Lamphear, B. J., Rhoads, R. E.,

17. Adamson, J. D., Herbert, E., and Hemp, S. F. (1969) J. Mol. Biol. 42,247-251 Kuechler, E., and Skern, T. (1993) Biochemistry 32, 7581-7588

18. Yan, R., Rychlik, W., Etchison, D., and Rhoads, R. E. (1992)J. Biol. Chem. 267,

20. Laemmli, U. K. (1970) Nature 227,680-685 19. Lamphear, B. J., and Panniers, R. (1990) J. Biol. Chem. 286,5333-5336

21. Morrisey, J. H11981)Anal. Eiochem. 117,307410 22. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, 1st Ed.,

23. Sommergruber, W., Ahorn, H., Zaphel, A., Maurer-Fogy, I., F e d , F., Schnor- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

renberg, G., Liebig, H.-D., Blaas, D., Kuechler, E., and Skern, T. (1992) J. B i d . Chem. 287,22639-22644

24. Etchison, D., Milburn, S. C., Edery, I., Sonenberg, N., and Hershey, J. W. B. (1982) J. Biol. Chem. 267, 14806-14810

25. Etchison, D., and Etchison, J. R. (1987) J. Virol. 81, 2702-2710 26. Etchison, D., Hansen, J., Ehrenfeld, E., Edery, I., Sonenberg, N., Milburn, S.,

27. Wyckoff, E. E., Hershey, J. W. B., and Ehrenfeld, E. (1990) Pmc. Natl. Acad.

28. Etchison, D., and Fout, S. (1985) J. Virol. 64, 634438 29. Duncan, R., Milburn, S. C., and Hershey, J. W. B. (1987) J. B i d . Chem. 282,

30. Skern, T., Sommergruber, W., Auer, H., Volkmann, P., Zorn, M., Liebig, H.-D.,

31. Skern, T., Sommergruber, W., Blaas, D., Gruendler, P., Frauendorfer, F., Pieler,

32. Jenkins, O., Booth, D., Minor, P., and Almond, J. (1987) J. Gen. Virol. 88,

Virology 160, 299-303

(1987) J. Virol. 82, 1243-1250

2322623231

and Hershey, J. W. B. (1984) J. Virol. 51, 832-837

Sci. U. S. A 87, 9529-9533

38&388

Fessl, F., Blaas, D., and Kuechler, E. (1991) Virology 181, 4 6 5 4

C., Fogy, I., and Kuechler, E. (1985) Nucleic Acids Res. 13, 2111-2126

1835-1848