Title: Caliciviruses Differ in Their Functional Requirements for eIF4F Components
Abstract: Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex. Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex. Translation initiation on eukaryotic mRNAs is a complex process, and many translational control mechanisms are focused on the initiation stage (1Pestova T.V. Kolupaeva V.G. Lomakin I.B. Pilipenko E.V. Shatsky I.N. Agol V.I. Hellen C.U. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7029-7036Crossref PubMed Scopus (603) Google Scholar, 2Gray N.K. Wickens M. Annu. Rev. Cell Dev. Biol. 1998; 14: 399-458Crossref PubMed Scopus (449) Google Scholar, 3Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (845) Google Scholar). The majority of host cell mRNAs are translated in a cap-dependent manner involving the recognition of their 5′ cap structure by the eIF4F initiation factor complex (4Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1766) Google Scholar). eIF4F is known as the "cap-binding complex" and comprises three proteins: (i) eIF4E, the only factor with direct cap-binding activity; (ii) eIF4A, an RNA helicase; and (iii) eIF4G, which functions as a scaffold to bind several other factors such as eIF3, poly(A)-binding protein (PABP), 6The abbreviations used are: PABP, poly(A)-binding protein; FCV, feline calicivirus; MNV, murine norovirus; IRES, internal ribosome entry site; LDV, Lordsdale virus; CRFK, Crandell-Rees feline kidney; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; FMDV, foot and mouth disease virus. eIF4E and eIF4A. Subsequent to eIF4F binding to the 5′ cap, the 43 S preinitiation complex is recruited to the mRNA via its interaction with eIF3 (5Kapp L.D. Lorsch J.R. Annu. Rev. Biochem. 2004; 73: 657-704Crossref PubMed Scopus (408) Google Scholar). Positive-stranded RNA viruses have evolved a variety of mechanisms for subverting the host cell translation machinery for their own use (6Belsham G.J. Jackson R.J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 869-900Google Scholar, 7Bushell M. Sarnow P. J. Cell Biol. 2002; 158: 395-399Crossref PubMed Scopus (147) Google Scholar). In many cases this results in the preferential translation of viral mRNAs in the presence of relatively high concentrations of competing host cell mRNAs. However, infection of cells by many picornaviruses also leads to the inhibition of host cell (cap-dependent) translation. This is primarily achieved through the cleavage of eIF4G, for example by the poliovirus 2A protease (8Krausslich H.G. Nicklin M.J. Toyoda H. Etchison D. Wimmer E. J. Virol. 1987; 61: 2711-2718Crossref PubMed Google Scholar, 9Lloyd R.E. Grubman M.J. Ehrenfeld E. J. Virol. 1988; 62: 4216-4223Crossref PubMed Google Scholar) or foot-and-mouth disease virus L protease (10Devaney M.A. Vakharia V.N. Lloyd R.E. Ehrenfeld E. Grubman M.J. J. Virol. 1988; 62: 4407-4409Crossref PubMed Google Scholar), resulting in the separation of the eIF4A- and eIF4E-binding sites. Picornavirus mRNAs are still translated because of the presence of an internal ribosome entry site (IRES) element in the 5′-untranslated region that directs a cap-independent mechanism of translation. The C-terminal cleavage fragment of eIF4G is generally sufficient to support picornavirus IRES function (6Belsham G.J. Jackson R.J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 869-900Google Scholar). Caliciviruses are a major cause of viral gastroenteritis and have been associated with over 85% of nonbacterial gastroenteritis outbreaks in Europe between 1995 and 2000 (11Lopman B.A. Reacher M.H. Van Duijnhoven Y. Hanon F.X. Brown D. Koopmans M. Emerg. Infect. Dis. 2003; 9: 90-96Crossref PubMed Scopus (273) Google Scholar). The human caliciviruses, including the prototype Norwalk virus, have yet to be fully propagated in tissue culture, although recent results suggest that limited genome replication and encapsidation can occur using a vaccinia virus-driven expression system (12Asanaka M. Atmar R.L. Ruvolo V. Crawford S.E. Neill F.H. Estes M.K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10327-10332Crossref PubMed Scopus (94) Google Scholar). In contrast to the human caliciviruses, feline calicivirus (FCV), porcine enteric calicivirus (13Chang K.O. Kim Y. Green K.Y. Saif L.J. Virology. 2002; 304: 302-310Crossref PubMed Scopus (29) Google Scholar, 14Chang K.O. Sosnovtsev S.V. Belliot G. Kim Y. Saif L.J. Green K.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8733-8738Crossref PubMed Scopus (115) Google Scholar), and murine norovirus 1 (MNV) (15Wobus C.E. Karst S.M. Thackray L.B. Chang K.O. Sosnovtsev S.V. Belliot G. Krug A. Mackenzie J.M. Green K.Y. Virgin H.W. PLoS Biol. 2004; 2: e432Crossref PubMed Scopus (687) Google Scholar) can be propagated in tissue culture. Reverse genetics systems also exist for both FCV (16Sosnovtsev S. Green K.Y. Virology. 1995; 210: 383-390Crossref PubMed Scopus (131) Google Scholar) and porcine enteric calicivirus (17Chang K.-O. Sosnovtsev S.S. Belliot G. Wang Q. Saif L.J. Green K.Y. J. Virol. 2005; 79: 1409-1416Crossref PubMed Scopus (62) Google Scholar). Therefore FCV, porcine enteric calicivirus, and MNV have been used as model systems to study calicivirus biology. We and others have previously reported that caliciviruses use a novel protein-directed translation initiation mechanism that involves the binding of translation initiation factors to the VPg protein that is covalently linked to the 5′ end of the viral RNA (18Daughenbaugh K.F. Fraser C.S. Hershey J.W. Hardy M.E. EMBO J. 2003; 22: 2852-2859Crossref PubMed Scopus (161) Google Scholar, 19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar). This mechanism has not been demonstrated in any other animal RNA virus but shares some similarity with a mechanism proposed for members of the plant potyvirus family (20Leonard S. Viel C. Beauchemin C. Daigneault N. Fortin M.G. Laliberte J.F. J. Gen. Virol. 2004; 85: 1055-1063Crossref PubMed Scopus (121) Google Scholar, 21Leonard S. Chisholm J. Laliberte J.F. Sanfacon H. J. Gen. Virol. 2002; 83: 2085-2089Crossref PubMed Scopus (29) Google Scholar, 22Leonard S. Plante D. Wittmann S. Daigneault N. Fortin M.G. Laliberte J.F. J. Virol. 2000; 74: 7730-7737Crossref PubMed Scopus (246) Google Scholar, 23Wittmann S. Chatel H. Fortin M.G. Laliberte J.F. Virology. 1997; 234: 84-92Crossref PubMed Scopus (222) Google Scholar). In our previous studies, we demonstrated that the VPg proteins of both FCV and Lordsdale virus (LDV), a human norovirus, interact directly with the eIF4E component of the eIF4F complex (19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar). Translation of FCV VPg-linked mRNA was blocked by the eIF4E inhibitor protein, 4E-BP1, confirming a functional role for eIF4E and suggesting that the eIF4E-4G interaction is essential for FCV translation. Because of the lack of a suitable source of VPg-linked NV RNA, we were unable to extend this observation to norovirus translation. In the current study we have used the recently identified MNV (24Karst S.M. Wobus C.E. Lay M. Davidson J. Virgin H.W.T. Science. 2003; 299: 1575-1578Crossref PubMed Scopus (658) Google Scholar) as a model for human norovirus translation. We have also extended our previous work on FCV to include the analysis of the other components of the eIF4F complex, namely eIF4G and eIF4A. We now demonstrate that the RNAs from caliciviruses differ in their functional requirements for the components of the eIF4F complex. Whereas each of the eIF4F factors are required for efficient translation of FCV mRNA, only eIF4A appears to play a critical role in MNV mRNA translation. Materials—FCV, strain Urbana, was generated from the full-length infectious clone pQ14 (16Sosnovtsev S. Green K.Y. Virology. 1995; 210: 383-390Crossref PubMed Scopus (131) Google Scholar) and propagated in Crandell-Rees feline kidney (CRFK) cells. MNV-1, strain CW.1, was the generous gift of Herbert Virgin (Washington University, St. Louis, MO) and was propagated in the murine macrophage cell line RAW 264.7 as described (15Wobus C.E. Karst S.M. Thackray L.B. Chang K.O. Sosnovtsev S.V. Belliot G. Krug A. Mackenzie J.M. Green K.Y. Virgin H.W. PLoS Biol. 2004; 2: e432Crossref PubMed Scopus (687) Google Scholar). Antisera to LDV VPg was generated by immunization of New Zealand White rabbits with recombinant VPg, purified as previously described (19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar). Antiserum to eIF4G was the kind gift of Simon Morley (University of Sussex). Antiserum to 4E-BP1 was purchased from Santa Cruz Biotechnology. Hippuristanol was isolated and used as described (25Bordeleau M. Mori A. Oberer M. Lindqvist L. Chard L.S. Higa T. Belsham G.J. Wagner G. Tanaka J. Pelletier J. Nat. Chem. Biol. 2006; 2: 213-220Crossref PubMed Scopus (278) Google Scholar). Expression and Purification of Recombinant Proteins—FCV VPg was expressed and purified as previously described (19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar). The cDNA encoding MNV VPg was PCR-amplified from a full-length clone of MNV kindly provided by Herbert Virgin (Washington University) using the primers IGRDG108 (5′-GCGCCGCGGTGGAGGAAAGAAGGGCAAGAACAAGAAGGGC) and IGRDG109 (5′-CGCGGATCCTTCTTCAGCAAAGCTAAC) and cloned into pET26Ub after digestion with SacII and BamHI. MNV VPg was purified from Escherichia coli by affinity chromatography on NiCam-agarose (Sigma), followed by an additional purification step on a heparin-Sepharose column (GE Healthcare). Recombinant 4E-BP1 was the generous gift of Simon Morley (University of Sussex). The eIF4A dominant negative mutant, DQAD (26Pause A. Methot N. Svitkin Y. Merrick W.C. Sonenberg N. EMBO J. 1994; 13: 1205-1215Crossref PubMed Scopus (327) Google Scholar), was expressed and purified as described (27Chard L.S. Kaku Y. Jones B. Nayak A. Belsham G.J. J. Virol. 2006; 80: 1271-1279Crossref PubMed Scopus (45) Google Scholar). Recombinant FMDV Lb protease was the generous gift of Tim Skern (University of Vienna). The eIF4E expression plasmid was the generous gift of Stephen Curry (Imperial College London). Histidine-tagged eIF4E was purified by affinity chromatography on a Hitrap chelating column (GE Healthcare) followed by cap-Sepharose (GE Healthcare). eIF4E was eluted from the cap-Sepharose column using a salt gradient to avoid possible contamination with cap analogue. Isolation of Calicivirus VPg-linked RNA from Infected Cells—FCV VPg-linked RNA was prepared from replication complexes isolated 4 h post-infection using the Genelute purification system (Sigma) as previously described (19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar). MNV VPg-linked RNA was prepared from RAW 264.7 cells, infected at a multiplicity of infection of 2 TCID50/cell, at 18 h post-infection, using the same system. Detection of VPg in MNV VPg-linked RNA Preparations—RNA isolated from either mock infected or MNV-infected cells was digested with RNase mixture (Ambion) for 1 h at 37°C prior to analysis by Western blotting with affinity purified anti-MNV VPg antiserum. In Vitro Translation Reactions—In vitro translation reactions were performed using the Flexi rabbit reticulocyte lysate system (Promega), using 25, 50, and 12.5 μg/ml of FCV, MNV, and control in vitro transcribed RNAs, respectively. These concentrations of RNA were previously determined to give a linear yield of translated product over the time course of the translation (90 min). In reactions that required the addition of either recombinant 4E-BP1, eIF4E, or the dominant negative mutant (DQAD) form of eIF4A, the reactions were preincubated with recombinant protein at 30 °C for 15 min prior to the addition of RNA. After 90 min, the reactions were terminated by the addition of SDS-PAGE sample buffer and subsequently resolved on 12.5% polyacrylamide gels. Pretreatment of RNA with proteinase K was carried out by incubation of RNA in 10 mm Tris, pH 8.0, 0.1 mm EDTA, 10 μg/ml proteinase K for 30 min at 37 °C followed by extraction with phenol:chloroform and precipitation with ethanol. MNV RNA was also treated as above but with the omission of proteinase K or the addition of EDTA-free protease inhibitor mixture (Roche Applied Science) as additional controls. Control in vitro transcribed RNA (of the form cap-CAT:IRES-Luc) (28Pisarev A.V. Chard L.S. Kaku Y. Johns H.L. Shatsky I.N. Belsham G.J. J. Virol. 2004; 78: 4487-4497Crossref PubMed Scopus (93) Google Scholar) was treated in the same manner. In reactions that contained exogenous cap analogue, 50 or 500 μm cap analogue (m7G(5′)ppp(5′)G, Promega) was preincubated with reticulocyte lysate at 30 °C for 15 min prior to the addition of RNA template. eIF4E Depletion—eIF4E was depleted from RRL as previously described (29McKendrick L. Morley S.J. Pain V.M. Jagus R. Joshi B. Eur. J. Biochem. 2001; 268: 5375-5385Crossref PubMed Scopus (76) Google Scholar). Briefly, 4E-BP1 (400 nm) was incubated with RRL at 30 °C for 10 min prior to the addition of 0.5 volume of cap-Sepharose. Bound complexes were removed by centrifugation, and the depleted lysate was aliquoted and frozen at –80 °C until required. In Vitro Transcription—Capped dicistronic mRNA was prepared from XhoI linearized pGEM-CAT:IRES-Luc (28Pisarev A.V. Chard L.S. Kaku Y. Johns H.L. Shatsky I.N. Belsham G.J. J. Virol. 2004; 78: 4487-4497Crossref PubMed Scopus (93) Google Scholar) or pGEM-rLuc:IRES-fLuc in which the CAT coding region was replaced with Renilla luciferase, using the Megascript transcription system (Ambion) in the presence of cap analogue (Promega). RNA was purified by lithium chloride precipitation and quantified by spectrophotometry. eIF4E Capture ELISA—Capture ELISAs to detect the interaction of eIF4E with VPg were performed essentially as described previously (22Leonard S. Plante D. Wittmann S. Daigneault N. Fortin M.G. Laliberte J.F. J. Virol. 2000; 74: 7730-7737Crossref PubMed Scopus (246) Google Scholar) except that murine eIF4E-GST or GST alone were purified using glutathione-Sepharose chromatography (GE Healthcare). The interaction of initiation factors with VPg was detected using an anti-GST monoclonal antibody. Cap-Sepharose Chromatography—Lysates from uninfected or infected RAW 264.7 cells, prepared 18 h post-infection with MNV, were incubated with cap-Sepharose (GE Healthcare) and eIF4E-containing complexes isolated as previously described (30Ptushkina M. von der Haar T. Karim M.M. Hughes J.M. McCarthy J.E. EMBO J. 1999; 18: 4068-4075Crossref PubMed Scopus (105) Google Scholar). Bound proteins were eluted with SDS-PAGE sample buffer and analyzed by Western blot. Virus Yield Assays—CRFK or RAW 264.7 cells were pretreated with hippuristanol (125 nm to 1 μm) or Me2SO as a control, for 1 h prior to infection with FCV or MNV (multiplicity of infection of 2). Infections were carried out in the presence of inhibitor/Me2SO for 30 min at room temperature, after which virus and inhibitor were removed, and the cells were washed with Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The cells were then incubated in Dulbecco's modified Eagle's medium with 10% fetal calf serum containing hippuristanol or Me2SO at 37 °C for 6 or 18 h for FCV and MNV, respectively. After two freeze/thaw cycles, the virus yield was determined by TCID50 on CRFK and RAW 264.7 cells for FCV and MNV, respectively. Cell Viability Assay—The effect of hippuristanol on the viability of CRFK and RAW 264.7 cells was examined using the CellTitre-Blue system (Promega) according to the manufacturer's instructions. CRFK and RAW 264.7 cells were incubated with various concentrations of inhibitor or Me2SO for 4 and 16 h, respectively. CellTitre-Blue reagent was then added to each well, and incubation continued for a further 2 h, after which the level of fluorescence was determined according to the manufacturer's instructions. Murine Norovirus RNA Translation Is Insensitive to Cap Analogue—To date, the study of norovirus translation has been limited to in vitro binding analysis of the recombinant norovirus VPg with translation initiation factors (18Daughenbaugh K.F. Fraser C.S. Hershey J.W. Hardy M.E. EMBO J. 2003; 22: 2852-2859Crossref PubMed Scopus (161) Google Scholar). To fully evaluate the functional roles of initiation factors in norovirus translation, an in vitro translation system was required. MNV has previously been shown to replicate efficiently in the STAT1-negative murine macrophage cell line RAW 264.7 (15Wobus C.E. Karst S.M. Thackray L.B. Chang K.O. Sosnovtsev S.V. Belliot G. Krug A. Mackenzie J.M. Green K.Y. Virgin H.W. PLoS Biol. 2004; 2: e432Crossref PubMed Scopus (687) Google Scholar), hence this offered a potential source of norovirus VPg-linked RNA. The MNV VPg-linked RNA was prepared by extracting total RNA from RAW 264.7 cells at various times post-infection. RNA prepared in a similar manner to FCV-infected cells was previously found to translate efficiently in the rabbit reticulocyte lysate system (19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar). Using similar in vitro translation conditions, RNA prepared from MNV-infected cells also translated efficiently in rabbit reticulocyte lysates (Fig. 1A). The translation profile of the RNA isolated at 6 or 18 h post-infection varied (Fig. 1A). Translation of a host cell mRNA present in uninfected extracts, highlighted in Fig. 1A with an asterisk, diminished during the course of infection and was absent when RNA was prepared from cells 18 h post-infection. However, the synthesis of some virus encoded products (e.g. 32-kDa product) was greatly enhanced using the RNA isolated at 18 h post-infection compared with the RNA isolated at 6 h. As we previously observed with FCV VPg-linked RNA, translation of MNV mRNA was found to require a protein covalently linked to the viral RNA (VPg), because pretreatment of the RNA with proteinase K ablated translation (Fig. 1B). The inclusion of protease inhibitors in the reaction prevented the effect of proteinase K (Fig. 1B). In contrast, translation of in vitro synthesized, control capped dicistronic RNA was unaffected by proteinase K pretreatment (Fig. 1B). We and others have previously demonstrated that FCV translation was insensitive to the addition of exogenous cap analogue (m7G(5′)ppp(5′)G) (19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar, 31Herbert T.P. Brierley I. Brown T.D. J. Gen. Virol. 1997; 78: 1033-1040Crossref PubMed Scopus (115) Google Scholar). To determine whether MNV mRNA translation also occurred in a cap analogue-insensitive manner, the effect of cap analogue on MNV mRNA in vitro translation was examined. Cap analogue had no significant effect on translation of MNV VPg-linked mRNA (Fig. 1C). Cap-dependent translation from in vitro synthesized control dicistronic was inhibited, whereas cap-independent translation from the FMDV IRES was unaffected (Fig. 1D). Further evidence that the observed in vitro translation profile was the result of VPg-dependent translation was the observation that RNA prepared in this manner was found to be infectious after transfection into permissive cells but noninfectious after treatment with proteinase K (data not shown). MNV VPg Interacts with eIF4E—We have previously demonstrated a direct interaction of the FCV and LDV VPg proteins with eIF4E in vitro (19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar). This interaction was also observed in infected cells because FCV VPg could be isolated using a cap-Sepharose affinity resin. To determine whether a similar interaction existed between MNV VPg and eIF4E, recombinant MNV VPg was expressed and purified from E. coli (Fig. 2A). A capture ELISA, whereby VPg from both FCV and MNV were the immobilized ligand, was performed to study the interaction with eIF4E (Fig. 2B). Recombinant GST-murine eIF4E was retained by both MNV and FCV VPg, whereas a control protein, maltose-binding protein, failed to bind detectable levels of eIF4E (Fig. 2B). GST alone did not bind to FCV VPg, MNV VPg, or 4E-BP1 (Fig. 2B). To confirm that the MNV VPg protein also interacted with eIF4E during infection, eIF4E containing complexes were isolated from infected cells via cap-Sepharose chromatography (Fig. 2C). Of the five forms of VPg generated during infection, all were retained on cap-Sepharose; however, the mature form and the ∼32-kDa precursor appear to be enriched (Fig. 2C). The levels of the eIF4F components present in cells or isolated by cap-Sepharose chromatography were unaffected by infection (Fig. 2C). Mature VPg, VPg precursors, or eIF4F components were not isolated on control Sepharose 4B alone (Fig. 2C). Glyceraldehyde-3-phosphate dehydrogenase was also not retained by cap-Sepharose, confirming the specificity of the assay (Fig. 2C). Only the Mature Form of VPg Is Linked to Viral RNA—Given our observation that all forms of VPg can be isolated by cap-Sepharose chromatography, we wished to examine the form of VPg found covalently linked to viral RNA. RNA isolated from either mock infected or MNV-infected cells was digested with RNase and subsequently analyzed by Western blot. In agreement with previous findings with FCV (31Herbert T.P. Brierley I. Brown T.D. J. Gen. Virol. 1997; 78: 1033-1040Crossref PubMed Scopus (115) Google Scholar), only the mature form of VPg was found to be covalently linked to MNV RNA (Fig. 2D). Translation of MNV RNA Is Not Inhibited by 4E-BP1—Our previous analysis revealed that in vitro translation of VPg-linked FCV mRNA was sensitive to the addition of the eIF4E repressor protein 4E-BP1 (19Goodfellow I. Chaudhry Y. Gioldasi I. Gerondopoulos A. Natoni A. Labrie L. Lailiberte J. Roberts L. EMBO Reports. 2005; 6: 968-972Crossref PubMed Scopus (166) Google Scholar). This protein binds to eIF4E and prevents the interaction with eIF4G (4Gingras A.C. Raught B. Sonenberg N. Annu. Rev. Biochem. 1999; 68: 913-963Crossref PubMed Scopus (1766) Google Scholar). These data would therefore suggest that the eIF4E-4G interaction is required for FCV mRNA translation. To determine whether a similar interaction is required for MNV mRNA translation, the effect of recombinant 4E-BP1 was examined (Fig. 3, A and B). Whereas recombinant 4E-BP1 inhibited FCV and cap-dependent translation from a control dicistronic mRNA as expected, MNV mRNA translation and FMDV IRES-directed translation were unaffected (Fig. 3, A and B). A protein translated from the MNV VPg-linked RNA preparations was also found to be sensitive to 4E-BP1 addition (Fig. 3A, asterisk). This is likely to represent a host cell mRNA that is translated in a cap-dependent manner and, where visible, will be referred to as CPX. Depletion of eIF4E Differentially Affects Calicivirus Translation—To further determine whether the MNV VPg-eIF4E interaction plays a significant role in MNV translation initiation, the translation of calicivirus RNA was examined in RRL depleted of eIF4E (Fig. 3C). eIF4E was depleted as described (29McKendrick L. Morley S.J. Pain V.M. Jagus R. Joshi B. Eur. J. Biochem. 2001; 268: 5375-5385Crossref PubMed Scopus (76) Google Scholar), and the concomitant removal of eIF4G was prevented by the prior addition of 4E-BP1. The eIF4E:4E-BP1 was subsequently removed by cap-Sepharose. Western blot analysis of depleted lysates demonstrated that whereas eIF4E levels were typically less than 10% of the levels seen in mock depleted lysates, the levels of the other eIF4F components, eIF4A and eIF4G, remained largely unaltered (Fig. 3D). Analysis of the levels of recombinant 4E-BP1 remaining in the depleted extract revealed that greater than 90% of the 4E-BP1 added to the reaction was removed during depletion (Fig. 3D). Note that the rabbit polyclonal antiserum used to detect the recombinant 4E-BP1 did not detect the endogenous 4E-BP1 present in the RRL (Fig. 3D). eIF4E depletion was found to specifically inhibit cap and FCV VPg-dependent translation, whereas FMDV IRES-mediated translation was largely unaffected (Fig. 3C). The addition of recombinant His-tagged eIF4E to a final concentration of ∼0.6 μm restored cap and FCV VPg-dependent translation to the levels found in mock depleted lysates (Fig. 3C). eIF4E depletion reduced MNV VPg-dependent translation to between 55 and 70% of the levels observed in mock depleted lysates (Fig. 3C). However, the addition of recombinant eIF4E to the depleted lysates did not restore MNV translation, suggesting that the observed effect was not due to the removal of eIF4E. FCV and MNV RNAs Differ in Their Requirements for Intact Full-length eIF4G—eIF4G plays several critical roles in translation initiation and is recruited to the 5′ end of mRNA via its interaction with eIF4E (32Pestova T.V. Kolupaeva V.G. Genes Dev. 2002; 16: 2906-2922Crossref PubMed Scopus (410) Google Scholar). To confirm that the eIF4E-eIF4G interaction was not required for MNV translation, the eIF4E-interacting domain of eIF4G was separated from the C terminus of eIF4G by cleavage with FMDV Lb protease, and the effect on calicivirus translation was analyzed (Fig. 4). Prior incubation of rabbit reticulocyte lysates with recombinant Lb protease led to a dose-dependent cleavage of eIF4G (Fig. 4A). As expected, eIF4G cleavage resulted in inhibition of cap-dependent translation, whereas FMDV IRES-directed translation was unaffected (Fig. 4, B and E). eIF4G cleavage resulted in inhibition of FCV mRNA translation but resulted in a slight stimulation of MNV translation (Fig. 4, C–E) probably because of a decrease in competition from cellular mRNAs or release of eIF4G from the eIF4E·eIF4G complex. A protein translated from MNV RNA preparations, described above as CPX (Fig. 4D, asterisk), was inhibited in reactions where eIF4G was cleaved with Lb protease (Fig. 4, D and E). Both MNV and FCV RNAs Require eIF4A for Translation