Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
Correspondence
Amelia Nieto
anmartin{at}cnb.uam.es
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ABSTRACT |
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Idoia Burgui and Tomás Aragón contributed equally to this work.
Present address: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.
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INTRODUCTION |
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Influenza virus mRNAs have a capped 5' end followed by a 1012 nt long untranslated region of cellular, heterogeneous sequences generated by cap-snatching, which precede a viral-encoded, highly conserved sequence that is common to all influenza virus genes. The 3' end of the viral mRNAs is polyadenylated by a reiterative copy of a U57 track present near the 5' end of the viral RNA (Luo et al., 1991; Poon et al., 1998
, 1999
; Robertson et al., 1981
). Although viral mRNAs are formally equivalent to cellular ones, influenza virus infection specifically enhances viral mRNA translation, with the conserved sequences contained within the 5'-untranslated region (5'UTR) playing a critical role (Garfinkel & Katze, 1993
). Recently, a specific interaction of the cellular RNA-binding protein GRSF-1 with the 5'UTR of the viral nucleoprotein mRNA has been described. This protein specifically stimulates nucleoprotein mRNA translation in HeLa cell extracts (Park et al., 1999
). In addition, viral mRNAs are preserved from the generalized degradation of cytoplasmic mRNAs that takes place in the course of infection (Beloso et al., 1992
; Inglis, 1982
).
NS1 protein is the only non-structural protein of influenza virus (Barret et al., 1979; Lamb & Choppin, 1979
). It accumulates in the nucleus of the infected cell at early times but is also present in the cytoplasm later in the infection, where it is associated with polysomes (de la Luna et al., 1995
; Falcón et al., 1999
; Krug & Etkind, 1973
). In view of its interaction with several viral and cellular factors, NS1 has been implicated in many of the alterations indicated above that occur during influenza virus infection. It recognizes double-stranded RNA, U6 snRNA, poly(A), viral vRNA and viral mRNA (Fortes et al., 1994
; Hatada & Fukuda, 1992
; Hatada et al., 1997
; Marión et al., 1997b
; Qiu & Krug, 1994
, 1995
), as well as the 30 kDa subunit of the CPSF complex, PABPII and NS1-BP, a nuclear protein that might be involved in splicing (Chen & Krug, 1999
; Nemeroff et al., 1998
; Wolff et al., 1998
). NS1 has anti-interferon properties. The role of its double-stranded RNA-binding domain in counteracting the antiviral pathways has been pointed out (Talon et al., 2000
; Wang et al., 2000b
). NS1 binding to the 30 kDa subunit of the CPSF complex has also been implicated as being responsible for its anti-interferon properties (Noah et al., 2003
).
NS1 is also involved in the stimulation of viral mRNA translation (de la Luna et al., 1995; Enami et al., 1994
; Katze et al., 1986
; Marión et al., 1997a
; Park & Katze, 1995
), a function in which its interaction with the 5'-terminal conserved sequences of viral mRNAs is important (de la Luna et al., 1995
; Park & Katze, 1995
). We have identified two cellular targets of NS1 that may be relevant for this function the human Staufen protein (Falcón et al., 1999
; Marión et al., 1999
) and the eIF4GI subunit of the eIF4F translation initiation factor (Aragón et al., 2000
). Mapping of the eIF4GI-binding domain in the NS1 protein has indicated that the first 113 N-terminal amino acids of the protein were sufficient to bind eIF4GI, but not the first N-terminal 81 residues. The first mutant has previously been shown to be a translational enhancer, while the second is defective in this activity (Marión et al., 1997a
). Since NS1 is able to bind poly(A) RNA (Qiu & Krug, 1994
) and its binding to eIF4GI protein takes place in a region located close to the poly(A)-binding protein (PABP1)-interacting domain (Aragón et al., 2000
), we considered the possibility that NS1 could replace PABP1 at the 3' poly(A) tail of viral mRNAs and, correspondingly, that an NS1eIF4GI interaction could impair PABP1eIF4GI association. In this report we show that NS1 associates specifically with viral mRNAs and does not compete with the eIF4GIPABP1 interaction in infected cells. On the contrary, NS1 is also able to interact with PABP1 in vivo and in vitro. Our data support a model for the preferential translation of viral mRNAs in which NS1 would promote their association to polysomes by interaction with eIF4GI and PABP1 proteins, as well as with the 5'-terminal conserved sequences of viral mRNAs.
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METHODS |
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Mutant construction.
The plasmid pGEX-2T-PABP1 expressed a GSTPABP1 fusion protein lacking the first nine amino acids of human PABP1. To obtain carboxy-deletion mutants of the GSTPABP1 protein, pGEX-2T-PABP1 plasmid was digested with HindIII or SpeI endonucleases, blunt-ended and self-ligated. The resulting plasmids pGEX-2T-PABP1 234 and pGEX-2T-PABP1 319 expressed a GSTPABP1 fusion protein containing the N-terminal 234 or 319 amino acids of PABP1, lacking the first 9 amino acids, fused to GST. To obtain amino-deletion mutants, pGEX-2T-PABP1 was digested with BseAI and BamHI, with BlpI and BamHI or with NcoI and BsrGI. In all cases the digestion mixture was blunt-ended and the fragments of interest were isolated and self-ligated. The resulting plasmids pGEX-2T-PABP1 1307, pGEX-2T-PABP1
1365 and pGEX-2T-PABP1
1535 expressed GSTPABP1 fusion proteins lacking 307, 365 or 535 amino acids at the N terminus, respectively.
Protein expression and purification.
The HisNS1 and HiseIF4GI 157550 proteins were purified as previously described (Aragón et al., 2000). GST protein, GSTPABP1 and its mutant derivatives were expressed in E. coli DH5 cells harbouring plasmids pGEX-2T, pGEX-2T-PABP1, pGEX-2T-PABP1
1307, pGEX-2T-PABP1 319, pGEX-2T-PABP1
1365, pGEX-2T-PABP1 234 and pGEX-2T-PABP1
1535. The proteins were purified on glutathioneSepharose according to the manufacturer's instructions (Pharmacia Biotechnology). Briefly, expression was induced with 1 mM IPTG for 2 h at 37 °C. The cells were resuspended in buffer containing 5 mM sodium phosphate, 150 mM NaCl, 1 % Triton X-100, 2 mM EDTA, pH 7·4 (supplemented before use with 1 mM PMSF, 1 mM TPCK, 1 mM TLCK and 0·1 % 2-mercaptoethanol) and sonicated. After removal of cell debris by centrifugation, the supernatant was incubated with glutathioneSepharose 4B resin equilibrated in the same buffer by rocking for 30 min at 4 °C. After extensive washes with the same buffer, the proteins were eluted with 10 mM glutathione in 50 mM Tris/HCl at pH 8·0.
In vitro transcription/translation.
Plasmids encoding NS1 protein or mutants thereof were used for in vitro transcription/translation using the Promega TNT coupled system. In all cases the genes were expressed under the T7 promoter and a 35S-labelled methionine/cysteine mixture (1400 µCi ml-1) was added to the cell-free protein synthesis system. After 2 h of incubation at 30 °C, the mixture was centrifuged at 10 000 g for 10 min at 4 °C and the supernatants centrifuged again at 250 000 g for 2 h at 4 °C. The post-ribosomal supernatants were then used as a source of recombinant protein for in vitro binding studies.
Western blotting.
This was done as described previously (Sanz-Ezquerro et al., 1995). The following primary antibodies were used: for eIF4GI, a mixture of rabbit antibodies against N-terminal or C-terminal peptides of eIF4GI (1 : 2000 dilution each) (Aragón et al., 2000
); for PABP1 protein, a rabbit antiserum raised against GSTPABP1 fusion protein (dilution 1 : 1000); for NS1 protein, a rabbit anti-NS1 serum prepared by hyperimmunization with HisNS1 protein (Marión et al., 1997a
) (1 : 300 dilution) or a rat anti-NS1 serum against HisNS1 protein (1 : 400); for His-tagged proteins, a rabbit anti-His peroxidase-conjugated serum (Santa Cruz Biotechnology) (1 : 10 000) dilution; and for GST-tagged proteins, a rabbit anti-GST serum (Sigma) (1 : 10 000) dilution.
Coimmunoprecipitation.
Cultures of COS-1 cells were mock-infected or infected with influenza virus A/Victoria/3/75 strain at an m.o.i. of 10. After the incubation time, the cells were washed with ice-cold PBS, scraped off the plates and lysed in a buffer containing 150 mM NaCl, 1·5 mM MgCl2, 10 mM Tris/HCl, pH 8·5 and 0·5 % Igepal (extraction buffer). The extracts were clarified by centrifugation at 10 000 g for 15 min and used for coimmunoprecipitation assays. The extracts were incubated with the corresponding antibody for 2 h at 4 °C and applied to protein ASepharose. When indicated, the extracts were treated with micrococcal nuclease for 15 min at 37 °C before the addition of the antibody. For Western blot assays, the immunoprecipitates were washed four times with extraction buffer, boiled in Laemmli sample buffer and analysed by SDS-PAGE. To analyse the RNA associated with NS1 protein, the immunoprecipitates from either mock-infected or influenza virus-infected cells were washed seven times with extraction buffer and twice with RIPA buffer (10 mM Tris/HCl, pH 7·5, 150 mM NaCl, 1 % sodium deoxycholate, 0·1 % SDS and 1 % Igepal) and the resulting protein ASepharoseIgG complexes were used to isolate the associated RNA as previously described (Marión et al., 1997b).
Pull-down experiments.
For pull-down experiments with GST fusion proteins, GST, GSTPABP1 or mutant GSTPABP1 proteins were purified as described above and bound to Sepharose 4Bglutathione resins. Purified HisNS1 or HiseIF4GI 157550, in vitro-translated NS1 or its deletion mutants were added and incubated for 1 h at room temperature in a buffer containing 150 mM NaCl, 10 mM Tris/HCl, pH 8·5, 1·5 mM MgCl2 and 0·5 % Igepal. After incubation, the resins were washed three times with 10 vols of the same buffer and the bound proteins were analysed by Western blot assays.
RNA analysis.
To identify the RNA associated with NS1 in vivo, the immunoprecipitates were extensively washed as described above. These immunoprecipitates or total cellular extracts prepared in 150 mM NaCl, 10 mM Tris/HCl, pH 8·5, 1·5 mM MgCl2 and 1 % Igepal were DNase treated as described (Ortín et al., 1980; Perales & Ortín, 1997
), incubated with 50 µg proteinase K ml-1 for 30 min at 37 °C and phenol extracted. The RNAs were heated for 15 min at 55 °C in 7·5 % formaldehyde in 10x SSC and applied to nylon filters. Duplicate filters were hybridized with negative-polarity riboprobes specific for the NP, M, vimentin or
-tubulin genes.
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RESULTS |
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The association of NS1 with eIF4GI (Aragón et al., 2000) could be the basis of the efficient translation of influenza virus mRNAs during infection. In line with this possibility, NS1 protein has been found to be associated with viral mRNAs (Marión et al., 1997a
). To ascertain the specificity of this association, we analysed the presence of viral and cellular mRNAs in NS1 immunoprecipitates at various times after infection with influenza virus. Cytosolic extracts from mock-infected or virus-infected cells were used to determine the accumulation of viral and cellular mRNAs by dot-blot hybridization. In addition, the extracts were used for immunoprecipitation with NS1-specific or control antibodies, and the presence in the immunoprecipitates of different cellular and viral mRNAs was studied. Owing to the degradation of cellular RNAs that occurs during influenza virus infection, dot-blot hybridization was used instead of Northern assays to evaluate the total amount of RNAs present in the preparations, since otherwise some hybridization signals could be lost. The results are presented in Fig. 1
. The total amount of influenza NP or M mRNA increased with progression of the infection (Fig. 1
, Total). The presence of NP and M mRNA associated with NS1 was clearly visible in the NS1-specific immunoprecipitates (Fig. 1
, Ipp Ab-NS1). In contrast, the mRNAs encoding
-tubulin and vimentin were not present in these immunoprecipitates. Neither NP nor M,
-tubulin or vimentin mRNAs were present in the control immunoprecipitates (Fig. 1
, Ipp Control). Quantitative determination of the hybridization signals from at least five independent experiments indicated that the ratio of mRNA present in the anti-NS1 versus control immunoprecipitates was around ten times higher for influenza mRNA than for cellular mRNA. These data indicate that during infection NS1 protein associates specifically with influenza virus mRNA.
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DISCUSSION |
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Rotaviruses have a segmented genome consisting of 11 molecules of double-stranded RNA. Their mRNAs are capped but non-polyadenylated (Estes & Cohen, 1989). The viral mRNAs have 5'- and 3'UTRs of variable length that are flanked by two different sequences common to all rotavirus genes. NSP3 is a non-structural protein that plays a critical role in translation regulation of rotavirus mRNAs. The protein can be cross-linked to the consensus sequence located at the 3' end and is coimmunoprecipitated with eIF4GI factor (Piron et al., 1998
). NSP3 interacts with the same region of eIF4GI that interacts with PABP1 (Piron et al., 1998
). As a consequence, during rotavirus infection, PABP1 dissociates from eIF4GI, probably impairing the translation of polyadenylated mRNA and leading to the cellular shutoff. The enhancement of viral mRNA translation by NSP3 has also been observed in vitro, and the phenotype of NSP3 mutants has shown that both its RNA- and eIF4GI-binding domains are required to enhance the translation of viral mRNAs (Vende et al., 2000
), indicating that interaction with these molecules is required to obtain a fully translational activation.
Picornaviruses and rotaviruses have both developed mechanisms to ensure efficient viral protein synthesis, concomitant with an impairment of endogenous protein translation. In both cases, an appropriate virus translation correlates with an efficient eIF4GIviral element interaction. These regulatory mechanisms involve changes in the normal composition of the translation machinery, cleavage of eIF4GI and PABP1 proteins during picornavirus infection or the release of the eIF4GI-bound PABP1 protein during rotavirus infection. These data suggest that modulation of these two factors or a coordinate regulation of both proteins could be sufficient to regulate translation. These changes finally give rise to a drastic reduction in the translation of cellular mRNAs that are 5' capped and 3' polyadenylated and require fully active translation initiation complexes.
NS1 associates with translation initiation factors
Influenza virus mRNAs are formally equivalent to the endogenous mRNAs and a different strategy has been selected by the virus to enhance specifically the translation of viral mRNAs. Previous work has demonstrated that NS1 protein interacts directly and specifically with eIF4GI factor in vitro and that they associate in vivo during influenza virus infection in what appears to be a feature common to other viruses to improve virus translation (Aragón et al., 2000). The results presented in this report indicate that NS1 and PABP1 also interact directly in vitro and are associated in the infection (Figs 3 and 4
). Mapping studies have shown that the interactions among NS1, eIF4GI and PABP1 are compatible. Thus, the eIF4GI- and PABP1-interacting domains map to positions 81113 and 181 of the NS1 protein, respectively. The PABP1-interacting domain in eIF4GI maps to position 132160 (Imataka et al., 1998
), whereas residues 157550 are involved in NS1 interaction (Aragón et al., 2000
). Finally, eIF4GI-binding domain is located in the region 1175 of PABP1 (Imataka et al., 1998
), while residues 365535 are required for NS1 interaction. Although NS1 also interacts with a different poly(A)-binding protein, PABII, amino acids 223237 of NS1 are required for this interaction. PABII is a nuclear protein required for the processive elongation of poly(A) chains catalysed by the poly(A) polymerase, which does not share sequence homology with the cytoplasmic PABP1 (Chen & Krug, 1999
; Nemeroff et al., 1998
; Li et al., 2001
). Sequence comparison has shown that PABP1 is very conserved among different species, from yeast to humans (Burd et al., 1991
). The highest homology is found in the RRMs and the last C-terminal 75 amino acids (more than 94 % identity). Several proteins interact at the C-terminal conserved region of PABP1. Among them, Paip1 stimulates translation (Craig et al., 1998
) and Paip2 competes with Paip1 for binding to PABP1 and represses translation (Khaleghpour et al., 2001
). The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) (Hoshino et al., 1999
) and the polymerase of zucchini yellow mosaic potyvirus (Wang et al., 2000a
) also bind to PABP1. Interestingly, NS1 binds to PABP1 in a non-conserved region different from that used by the other PABP1-interacting proteins (Roy et al., 2002
). The possibility exists that the NS1PABP1 interaction is restricted to species to which influenza virus has become adapted during evolution.
The interaction between eIF4GI and PABP1, which facilitates translation initiation of polyadenylated mRNAs in yeast and stimulates translation in Xenopus oocytes and mammalian cells (Gray et al., 2000; Imataka et al., 1998
; Sachs et al., 1986
; Tarun & Sachs, 1996
; Tarun et al., 1997
; Wakiyama et al., 2000
), may be reinforced by the cross-interaction of both factors with NS1 protein in influenza virus-infected cells. Furthermore, the immunoprecipitation of viral mRNAs, but not cellular mRNAs, with anti-NS1 antibodies (Fig. 1
) strongly supports previous data indicating a specific interaction of NS1 with viral mRNA (Katze et al., 1986
; Marión et al., 1997b
; Park & Katze, 1995
) and suggests an NS1-mediated enhancement of virus translation. Virus-specific mRNAs would be efficiently associated with translation initiation complexes by the concurrent interaction of NS1 protein with their 5' end as well as with eIF4GI and PABP1. These complexes would effectively recruit ribosomes to the 5' end of viral mRNAs at the expense of cellular ones.
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ACKNOWLEDGEMENTS |
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Received 7 July 2002;
accepted 19 August 2003.