Cellular cap-binding proteins associate with influenza virus mrnas

Journal of General Virology (2011), 92, 1627 1634 DOI 10.1099/vir.0.029231-0 Cellular cap-binding proteins associate with influenza virus mrnas Katja Bier, Ashley York and Ervin Fodor Correspondence Ervin Fodor ervin.fodor@path.ox.ac.uk Received 25 November 2010 Accepted 6 March 2011 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK The influenza virus RNA polymerase synthesizes three types of RNA: genomic vrna, antigenomic crna and mrna. Both vrna and crna are bound by the viral RNA polymerase and nucleoprotein to form ribonucleoprotein complexes. Viral mrnas are also proposed to be bound by the RNA polymerase to prevent their endonucleolytic cleavage, regulate the splicing of M1 mrna, and facilitate translation. Here, we used standard immunoprecipitation, biochemical purification and RNA immunoprecipitation assays to investigate the association of viral and host factors with viral mrna. We found that viral mrna associates with the viral non-structural protein 1 (NS1), cellular poly(a)-binding protein 1 (PABP1), the 20 kda subunit NCBP1 of the nuclear cap-binding complex (CBC), the RNA and export factor-binding protein REF/Aly and the translation initiation factor eif4e. However, our data suggest that the RNA polymerase might not form part of the viral messenger ribonucleoprotein (mrnp) complex. We propose a model in which viral mrnas, by associating with cellular cap-binding proteins, follow the pathways normally used by cellular mrnas for splicing, nuclear export and translation. INTRODUCTION Influenza A virus is a negative-sense RNA virus containing eight segments of ssrna as its genome. During the viral life cycle, the RNA genome is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp) complex that consists of three subunits, PB1, PB2 and PA. The PB1 subunit acts as the polymerase by catalysing RNA chain elongation, while PB2 and PA are involved in the generation of capped RNA primers for the initiation of viral transcription. PB2 binds to the 59 cap of host mrnas, which are subsequently cleaved by the PA subunit approximately 10 15 nt downstream of the cap structure to generate primers that are elongated by PB1 to produce viral mrna. Viral mrnas are polyadenylated by stuttering of the viral RdRp on a sequence of uridine residues near the 59 end of the vrna template. Thus, viral mrnas structurally resemble host mrnas by having a 59 cap and a 39 poly(a) tail. In contrast, anti-genomic crnas produced during viral replication are full-length copies of vrna lacking a 59 cap and a 39 poly(a) tail. They serve as templates for the synthesis of genomic vrna (reviewed by Neumann et al., 2004; Palese & Shaw, 2007; Ruigrok et al., 2010). While vrnas and crnas associate with the viral RdRp and multiple copies of the nucleoprotein to form ribonucleoprotein complexes, much less is known about the proteins that associate with the viral mrnas. Cellular mrnas form large messenger ribonucleoprotein (mrnp) complexes involving numerous proteins that participate in the Supplementary tables are available with the online version of this paper. processing, nuclear export and translation of mrnas. Assembly of cellular mrnps occurs co-transcriptionally and this is facilitated by the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) (reviewed by Aguilera, 2005). The viral RdRp also associates with the CTD of Pol II, suggesting a mechanism by which viral mrnas might access the pathways used by cellular mrnas (reviewed by Amorim & Digard, 2006; Engelhardt & Fodor, 2006). Indeed, viral mrnas associate with NXF1, a factor that is known to be involved in the nuclear export of cellular mrnas (Read & Digard, 2010; Wang et al., 2008). However, several reports suggest that in addition to host factors, viral factors also participate in viral mrnp assembly. In particular, the viral non-structural protein 1 (NS1) has been implicated in the regulation of the splicing of viral mrnas, their nucleo-cytoplasmic transport and the enhancement of their translation (reviewed by Hale et al., 2008; Schneider & Wolff, 2009). The viral RdRp is also believed to bind viral mrna through the 59 terminal cap and the conserved AGCAAA- GCAGG sequence, present in all viral mrnas, downstream of the host mrna-derived primer sequence (Shih & Krug, 1996). It was suggested that this interaction is likely to prevent the association of viral mrna with the nuclear cap-binding complex (CBC) that normally associates with the cap structure of cellular mrnas in the nucleus (Shih & Krug, 1996). The association of the viral RdRp with viral mrna was also proposed to prevent their endonucleolytic cleavage by the viral RdRp itself and to regulate the splicing of the M1 mrna (Shih & Krug, 1996; Shih et al., 1995). Further studies suggested that the RdRp might remain 029231 G 2011 SGM Printed in Great Britain 1627

K. Bier, A. York and E. Fodor associated with the mrna not only in the nucleus, but also during mrna export to the cytoplasm where the RdRp could functionally replace the translation initiation factor eif4e (Burgui et al., 2007). These studies strongly suggest that the RdRp is a stable component of the viral mrnps. However, while the first two studies analysed the association of purified viral RdRp with capped RNA in vitro, the latter study only investigated the association of the RdRp with the translation initiation complex, but not viral mrna directly. Here, we investigate the association of viral mrnas with viral and cellular factors further. Our results show that the viral RdRp is unlikely to form a stable component of viral mrnps. In contrast, the cellular cap-binding proteins NCBP2 and eif4e were found to co-immunoprecipitate viral mrnas, suggesting that they participate in the assembly of viral mrnps. RESULTS Viral mrna does not co-immunoprecipitate with the viral RdRp from infected cells To investigate the role of the influenza virus RdRp in the assembly of viral mrnps, we initially carried out a standard immunoprecipitation of the RdRp from 293T cells infected with influenza A/WSN/33 virus, followed by the analysis of co-immunoprecipitated RNAs by a primer extension assay. We found that a substantial fraction of the NA gene-specific vrna and crna was co-immunoprecipitated with a PAspecific antibody, while only very low levels of mrna could be detected (Fig. 1a). No 5S rrna could be detected in the immunoprecipitates, confirming the specificity of the detected interaction of the RdRp with vrna and crna. Similar results were obtained for the M gene-specific RNAs (Fig. 1a). Next, we performed immunoprecipitations with antibodies specific for NS1 and poly(a)-binding protein 1 (PABP1), factors previously implicated in viral mrna binding (Burgui et al., 2003; Marión et al., 1997), to address the question of the feasibility of co-immunoprecipitating viral mrna with a standard immunoprecipitation protocol. We found that as expected (Marión et al., 1997) the NS1- specific antibody co-immunoprecipitated viral mrna, as well as vrna and crna (Fig. 1b), while the PABP1-specific antibody immunoprecipitated only viral mrna (Fig. 1c). Neither of the antibodies co-immunoprecipitated 5S rrna (Fig. 1b, c), indicating that the observed interactions are specific. As the PABP1-specific antibody co-immunoprecipitated viral mrna very efficiently, we asked the question whether any viral RdRp co-immunoprecipitated with these complexes. Although PB2 and PA were present in the cell lysates, they could not be detected in the immunoprecipitates obtained with the PABP1-specific antibody (Fig. 1c). However, the NS1 protein that was found to associate with viral mrna (Fig. 1b) could also be detected in PABP1 immunoprecipitates (Fig. 1c), in agreement with a previous study (Burgui et al., 2003). Taken together, these results show that the viral NS1 protein and the host protein PABP1 interact with viral mrna and suggest that the viral RdRp may not stably associate with viral mrna. Viral mrna does not co-purify with viral RdRp from transfected cells It is possible, although unlikely, that the PA-specific polyclonal antibody used above would not have recognized Fig. 1. Association of viral RdRp and NS1 protein and cellular PABP1 with viral RNAs during infection. Immunoprecipitations of lysates from influenza virus-infected cells were performed by using antibodies specific for (a) PA (a gift of T. Toyoda), (b) NS1 (a gift of J. Ortín) and (c) PABP1 (Abcam). RNA was isolated from cell lysates or immunoprecipitates (IP) and analysed by NA (a c) and M (a) gene-specific primer extension. A primer specific for 5S rrna was used as a control in all analyses. (a) The band detected in the 5S rrna panel of IP samples (highlighted by *) does not co-migrate with 5S rrna in lysates and it most probably represents a primer extension product related to M vrna-derived degradation products. (a c) Cell lysates and immunoprecipitates were also analysed by Western blot for the presence of the proteins being immunoprecipitated. 1628 Journal of General Virology 92

Binding of viral and host factors to viral mrna the RdRp specifically associating with mrna due to the mrna masking the epitopes recognized by the antibody. To address this possibility, we used an alternative strategy. A C-terminally Strep-tagged PA polymerase subunit was co-expressed with PB1, PB2, NP and NA vrna to reconstitute ribonucleoprotein (RNP) complexes in transfected 293T cells. The Strep-tag did not interfere with PA function, in agreement with previous findings with a C-terminal GFP-tag (Fodor & Smith, 2004), as similar amounts of mrna, crna and vrna were detected both in the presence of untagged PA and PA-Strep (Fig. 2). To analyse which viral RNAs interact with the viral RdRp in RNP reconstitution, the RdRp was purified by using Strep-Tactin agarose and copurified RNAs were isolated. Primer extension analysis of NA gene-specific RNAs shows that if an untagged PA was used, only very low levels of mrna and vrna could be detected, possibly representing non-specific background. If PA-Strep was used, both crna and vrna were enriched, while no mrna was detected above the background observed with untagged PA. No 5S rrna was co-purified with the Strep-tagged RdRp. Thus, the Strep-tagged RdRp specifically interacts with vrna and crna, but not with viral mrna in a viral ribonucleoprotein reconstitution assay. Only small amounts of viral mrna associate with the viral RdRp in RNA immunoprecipitation (RIP) assay To address the possibility that the interaction between the viral RdRp and mrna is unstable and the complex Fig. 2. Association of viral RdRp with viral RNAs in ribonucleoprotein reconstitution assays. 293T cells were transfected with pcdna-pb1, pcdna-pb2, pcdna-np, ppoli-na-rt and pcdna-pa or pcdna-pa-strep plasmids as indicated to express the components of viral ribonucleoprotein complexes (Fodor et al., 2002). The Strep-tagged RdRp was purified and co-purified RNA was analysed by NA gene-specific primer extension. A primer specific for 5S rrna was used as a control. Cell lysates and purified fractions were also analysed by Western blot for the presence of PA. dissociates during the immunoprecipitation or purification procedures above, we used a RIP assay, a method analogous to chromatin immunoprecipitation (ChIP) that involves the stabilization of protein nucleic acid complexes by formaldehyde treatment. The viral RdRp was immunoprecipitated with a PA-specific antibody and immunoprecipitated NP and M gene-specific RNAs were analysed by primer extension (Fig. 3a). Although viral mrna, crna and vrna as well as 5S rrna could be detected in the cell lysates, only crna and vrna were present at significant levels in the immunoprecipitated samples. Quantitative data from RT-qPCR indicate that about 10 20 % of NP and M vrna and crna was immunoprecipitated at all time points analysed (Fig. 3b). However, only between 0.3 and 2.6 % of the NP and M1 mrnas could be immunoprecipitated, possibly representing nascent mrnas. Viral mrna associates with cellular cap-binding proteins The viral RdRp has been proposed to prevent the binding of nuclear CBC to viral mrna and functionally replace the translation initiation factor eif4e by binding to the 59 end of viral mrna (Burgui et al., 2007; Shih & Krug, 1996). As we found no evidence for the stable association of the viral RdRp with viral mrna, we analysed the association of viral mrna with cellular cap-binding proteins. 293T cells were infected with influenza A/WSN/33 virus and cell lysates were immunoprecipitated with antibodies specific for the cellular cap-binding proteins, followed by the analysis of co-immunoprecipitated RNAs by primer extension. We found that an antibody directed against the 20 kda subunit (NCBP2) of CBC specifically co-immunoprecipitated NA-specific viral mrna (Fig. 4a). Similar results were obtained for NP-specific mrna. CBC binds to the 59 cap structure of nascent mrnas and plays a role in recruiting factors required for splicing and nuclear export of mrnas. One of these factors is the RNA and export factor-binding protein REF/Aly, a component of the transcription/export complex (TREX) (Topisirovic et al., 2011). In order to address whether REF/Aly associates with viral mrna, immunoprecipitations of lysates from virusinfected cells were performed using a REF/Aly-specific antibody. REF/Aly was found to co-immunoprecipitate NA-specific viral mrna (Fig. 4b). After mrnp nuclear export, the nuclear CBC is replaced by the cytoplasmic capbinding protein eif4e, which recruits other components of the translation initiation complex to the 59 cap. We found that eif4e associates with both NA- and NP-specific viral mrnas (Fig. 4c). Note that only very low levels or no crna, vrna or 5S rrna co-immunoprecipitated with cellular cap-binding proteins and REF/Aly (Fig. 4a c). Taken together these results strongly suggest that influenza virus mrna, by interacting with the nuclear and cytoplasmic cap-binding proteins, follows the pathways used by host mrnas for splicing, nuclear export and translation. http://vir.sgmjournals.org 1629

K. Bier, A. York and E. Fodor Fig. 3. RIP analysis of the association of the viral RdRp with viral RNA during the course of an infection. 293T cells were infected with influenza A/WSN/33 virus for the indicated period of time or were mock infected (M) followed by RIP. (a) RNAs from cell lysates and immunoprecipitates (IP) were analysed by NP and M genespecific primer extension. A primer specific for 5S rrna was used as a control. Note that 10 times more sample was used to analyse the RNAs in the immunoprecipitates compared with the cell lysates. (b) NP and M vrna, crna and mrna from cell lysates and immunoprecipitates were analysed by RT-qPCR and the amount of immunoprecipitated RNA was expressed as a per cent of RNA in cell lysates. The results shown represent a mean of three independent RIP analyses with SD. DISCUSSION In this study, we investigated the association of influenza virus mrnas with viral and cellular factors. We found that the viral protein NS1 and cellular proteins PABP1, NCBP2, REF/Aly and eif4e interact with viral mrna, but found no evidence for a stable association of the viral RdRp with viral mrna. Thus, our data do not support previous models in which the viral RdRp replaces the CBC at the 59 end of viral mrna in the nucleus (Shih & Krug, 1996). Instead, we found that viral mrna co-immunoprecipitates with the 20 kda subunit of CBC, NCBP2 (Fig. 4a). These results suggest that once the 59 cap of viral mrna is released from the PB2 subunit of the transcribing RdRp (Braam et al., 1983), it might associate with the CBC that would prevent the binding of a free non-transcribing RdRp to the 59 terminal cap and the conserved viral sequences downstream of the primer (Fig. 5). In fact, at the initial stages of viral infection, no free RdRp would be present to bind viral mrna. The binding of CBC to viral mrna might be facilitated by the association of the viral RNA polymerase with the CTD of Pol II (Amorim & Digard, 2006; Engelhardt & Fodor, 2006). CBC itself is known to bind to the CTD of Pol II through its 80 kda subunit, NCBP1 (Topisirovic et al., 2011). It should be noted that relatively small amounts of viral mrna were coimmunoprecipitated with the nuclear CBC (Fig. 4a), which is consistent with the transient nature of the interaction with cellular mrnas (Topisirovic et al., 2011). Quantitative comparison of the association of CBC with viral and cellular mrnas would provide further information about the significance of this interaction. The CBC bound to the 59 cap structure might be responsible for mediating the recognition of viral mrna by various components of the nuclear RNA processing machinery (Topisirovic et al., 2011). Indeed, we found that REF/Aly that is recruited to cellular mrna through the NCBP1 subunit of CBC also interacts with viral mrna (Fig. 4b). REF/Aly is a component of the TREX complex that is responsible for bridging the interaction of mrna with nuclear export receptor NXF1/TAP (Topisirovic et al., 2011). NXF1/TAP has been shown to co-localize and interact with viral mrna and its depletion by sirna treatment resulted in the retention of viral mrna in the nucleus (Read & Digard, 2010; Wang et al., 2008). Indeed, NXF1/TAP has been identified as an important host factor required for influenza virus replication in several genome-wide RNAi screens (reviewed by Watanabe et al., 2010). Our data suggest that NXF1/TAP could be recruited to viral mrna by REF/Aly, which itself is recruited as part of the TREX complex by the 80 kda subunit of CBC. If the viral RdRp does not associate stably with viral mrna, the questions of how viral mrna could be protected from endonucleolytic cleavage and how the regulation of splicing of the M1 mrna is achieved arise. Although it was demonstrated that the RdRp can perform 1630 Journal of General Virology 92

Binding of viral and host factors to viral mrna Fig. 4. Association of cellular cap-binding proteins NCBP2 and eif4e and the RNA and export factor-binding protein REF/Aly with viral RNAs during viral infection. Immunoprecipitations of lysates from influenza virus-infected cells were performed by using antibodies specific for (a) NCBP2 (Abcam), (b) REF/Aly (a gift of R. Reed) and (c) eif4e (Abcam). RNA was isolated from cell lysates or immunoprecipitates (IP) and analysed by NA (a c) and NP (a, c) gene-specific primer extension. A primer specific for 5S rrna was used as a control in all analyses. Cell lysates and immunoprecipitates were also analysed by Western blot for the presence of the proteins being immunoprecipitated. these functions in vitro (Shih & Krug, 1996; Shih et al., 1995), we can only speculate that in vivo these functions are performed by alternative mechanisms not involving the RdRp. For example, the viral NS1 protein was found to regulate splicing (Fortes et al., 1994; Lu et al., 1994; Muraki et al., 2010) and in this study we confirmed previous findings that NS1 associates with viral mrna (Fig. 1b). Indeed, ribonucleoprotein reconstitution assays with the M gene suggest that the NS1 protein is involved in the regulation of splicing of the M1 mrna (N. Robb and E. Fodor, unpublished data). Alternatively, the splicing of M1 mrna could be controlled by the rate of nuclear export (Valcárcel et al., 1993). It should also be noted that we detected low levels of viral mrna associating with the viral RdRp when a RIP assay followed by a highly sensitive RTqPCR was used (Fig. 3b). Although we assume that these Fig. 5. Schematic representation of the association of influenza virus mrna with cellular cap-binding proteins. The viral RdRp associates with the C-terminal domain of cellular RNA polymerase II in order to access the 59 cap of nascent cellular transcripts (dashed line) (Amorim & Digard, 2006; Engelhardt & Fodor, 2006). The PB2 subunit binds to the 59 cap and the PA subunit performs endonucleolytic cleavage to generate a capped RNA primer (Dias et al., 2009; Fechter et al., 2003; Guilligay et al., 2008; Yuan et al., 2009). The cleaved cellular nascent mrna is degraded by host nucleases, leading to Pol II termination (Chan et al., 2006; West et al., 2004). The PB1 subunit initiates viral mrna (solid line) synthesis by extending the capped primer. Subsequently, the PB2 subunit releases the 59 cap (Braam et al., 1983) and the nuclear CBC is free to bind to it to initiate the process of packaging of viral mrna into mrnps, i.e. the binding of the RNA and export factor-binding protein REF/Aly and other factors (Topisirovic et al., 2011). After export into the cytoplasm, CBC is replaced by the cytoplasmic cap-binding protein, the translation initiation factor eif4e, which triggers the recruitment of further factors involved in translation (e.g. eif4g). eif4g associates with PABP1, which induces the circularization of viral mrna. Note that for simplicity the vrna template associated with the viral RdRp is not shown. http://vir.sgmjournals.org 1631

K. Bier, A. York and E. Fodor are most likely nascent transcripts that have not yet been released from the transcribing RdRp, we cannot completely exclude the possibility of some free RdRp binding to mrna and contributing to their maturation. It has also been proposed that the viral RdRp can functionally replace the translation initiation factor eif4e possibly by associating with the 59 end viral mrnas and recruiting the translation machinery to viral mrnas (Burgui et al., 2007). We found that viral mrnas coimmunoprecipitate with eif4e strongly suggesting that after nuclear export, CBC is replaced by the cytoplasmic eif4e (Fig. 5). eif4e could then initiate the recruitment of the remaining components of the translation initiation complex by binding to eif4g. eif4g binds PABP1, another factor found to be associated with viral mrnas [Fig. 1(c) and Burgui et al., 2003], possibly facilitating the circularization of mrna (Fig. 5 and reviewed by Topisirovic et al., 2011). However, influenza virus infection has been reported to proceed normally in different situations of functional impairment of the eif4e factor (Burgui et al., 2007), suggesting that eif4e might not be essential for the translation of influenza virus mrnas. Furthermore, NS1 has been found to co-immunoprecipitate viral mrnas (Fig. 1b) and interact with PABP1 and eif4g (Aragón et al., 2000; Burgui et al., 2003), possibly promoting the enhanced translation of viral mrnas (de la Luna et al., 1995; Enami et al., 1994; Park & Katze, 1995). However, the exact molecular mechanisms involved remain unknown. It should also be noted that viral mrna translation proceeds almost normally in IFN-deficient cells infected with an influenza virus lacking the NS1 gene, suggesting that although NS1 might have a regulatory function, it is clearly not essential (Salvatore et al., 2002). In summary, further studies will be required to fully understand the regulatory mechanisms involved in the splicing of viral mrnas, their assembly into mrnps, nuclear export and translation. Our studies suggest that the viral RdRp plays no significant role in these processes once the synthesis of the viral mrna is completed by polyadenylation by the viral RdRp (Poon et al., 1999) and the mrna transcript is released from the transcribing viral RdRp. The nuclear CBC is likely to bind to the 59 cap of viral mrnas, triggering the recruitment of cellular factors normally involved in mrnp assemblies, followed by the replacement of the nuclear CBC with the translation initiation factor eif4e in the cytoplasm. Thus, it is likely that influenza virus mrna follows the pathways normally used by cellular mrnas for splicing, nuclear export and translation. METHODS Cells and viruses. Human kidney 293T cells were obtained from the cell bank of the Sir William Dunn School of Pathology and were maintained in minimal essential medium containing 10 % FCS at 37 uc with 5 % CO 2. Influenza A/WSN/33 virus was provided by Peter Palese (Mount Sinai School of Medicine, New York, USA). Plasmids. The protein expression plasmids pcdna-pb1, pcdna- PB2, pcdna-pa and pcdna-np have been described previously (Fodor et al., 2002), as have the ppoli-na-rt, ppoli-np-rt and ppoli-m-rt vrna transcription plasmids (Fodor et al., 1999; Pleschka et al., 1996). The pcdna-pa-strep construct was generated by replacing the TAP-tag of pcdna-pa-tap (Deng et al., 2005) with the eight amino acids (WSHPQFEK) of the Strep-tag. Two complementary oligonucleotides (encoding the Strep-tag) 59-CTAG- ATTATTTTTCGAACTGCGGGTGGCTCCAGGC-39 and 59-CGGA- CCTCGGTGGGAGTCAAGCTTTTTATTAGATC-39 were annealed and cloned into NotI/XbaI-digested pcdna-pa-tap plasmid. Purification of Strep-tag fusion proteins. RNP reconstitution assays were carried out in 10 cm cell culture dishes by transfecting suspensions of 293T cells (approx. 6610 6 cells per dish) with 5 mg of each of ppoli-na-rt, pcdna-pb1, pcdna-pb2, pcdna-np and pcdna-pa or pcdna-pa-strep plasmids using 50 ml Turbofect transfection reagent (Fermentas) following the manufacturer s instructions. After 48 h, cells were harvested and pelleted by centrifugation, washed in PBS and lysed in 500 ml Tris-lysis buffer [50 mm Tris/HCl, ph 8.0, 25 % (v/v) glycerol, 0.5 % Igepal CA-630, 100 mm NaCl, 1 mm DTT, 1 mm PMSF and 1 complete mini EDTA-free protease inhibitor cocktail tablet (Roche) per 10 ml of buffer] for 1 h at 4 uc. The lysates were then clarified by centrifugation in a Fisher microcentrifuge at 13 000 r.p.m. for 5 min at 4 uc and stored at 220 uc. For the purification of Streptagged RNA polymerase, 150 ml cell lysates was incubated with 75 ml Strep-Tactin agarose (Novagen) for 2 3 h at 4 uc. After three washes in wash buffer (150 mm NaCl, 100 mm Tris/HCl ph 8.0, 1 mm EDTA) half of the sample was used for the analysis of purified proteins by heating the Strep-Tactin agarose for 5 min in 50 ml SDSprotein loading buffer before loading onto SDS-PAGE. The other half of the sample was used for the isolation of co-purified RNA by treating the Strep-Tactin agarose with 1 ml TRIzol reagent (Invitrogen) following the manufacturer s instructions. Precipitated RNA was dissolved in 10 ml nuclease-free water and stored at 220 uc. Standard immunoprecipitations. Semi-confluent monolayers of 293T cells in 10 cm cell culture dishes were infected with influenza A/WSN/33 virus at an m.o.i. of 5 in 1 ml MEM with 0.5 % FCS for 1 h at room temperature. The virus inoculum was aspirated and 10 ml MEM containing 0.5 % FCS was added, followed by incubation at 37 uc for 5 h. Cells were harvested by centrifugation, washed in PBS and lysed in 500 ml Tris-lysis buffer [50 mm Tris/HCl, ph 8.0, 33 % (v/v) glycerol, 0.5 % Igepal CA-630, 200 mm NaCl, 1 mm DTT, 1 mm EDTA and 1 complete mini EDTA-free protease inhibitor cocktail tablet (Roche) per 10 ml of buffer] for 1 h at 4 uc. The lysates were then clarified by centrifugation in a Fisher microcentrifuge at 13 000 r.p.m. for 5 min at 4 uc and stored at 220 uc. For immunoprecipitations, 100 ml cell lysates was incubated with the appropriate antibodies for 2 h at 4 uc. After a brief spin in a microcentrifuge, the supernatant was mixed with 6 mg protein A- Sepharose (Sigma) and the mixture was incubated for 2 h. After four washes in wash buffer (10 mm Tris/HCl, ph 8.0, 150 mm NaCl, 0.1 % Igepal CA-630, 1 mm EDTA, 1 mm PMSF) one-fifth of the sample was used for the analysis of immunoprecipitated proteins by heating the protein A-Sepharose for 5 min in 50 ml SDS-protein loading buffer before analysis by SDS-PAGE and Western blot. The remainder of the sample was used for the isolation of coimmunoprecipitated RNA by treating the protein A-Sepharose with 1 ml TRIzol reagent (Invitrogen) following the manufacturer s instructions. Precipitated RNA was dissolved in 50 ml nuclease-free water and stored at 220 uc. Immunoprecipitations involving formaldehyde-mediated crosslinking (RIP). RNA immunoprecipitations were performed by using the previously described ChIP protocol (Chan et al., 2006; Vreede et al., 1632 Journal of General Virology 92

Binding of viral and host factors to viral mrna 2010). Briefly, 293T cells were infected with influenza A/WSN/33 virus as for standard immunoprecipitations (see above) and the cells were treated with 1 % formaldehyde for 10 min to covalently cross-link interacting proteins and nucleic acids at 3, 6, 9 and 12 h post-infection. Cross-linking reactions were stopped by the addition of glycine to a final concentration of 125 mm. Cells were washed with PBS, resuspended in 500 ml cell lysis buffer [50 mm Tris/HCl, ph 8.0, 200 mm NaCl, 0.5 % Igepal CA-630, 1 mm DTT, 1 mm EDTA and 1 complete mini, EDTA-free protease inhibitor cocktail tablet (Roche) per 10 ml of buffer] and were sonicated in a Bioruptor (Diagenode) for 15 min. Clarified cell lysates (100 ml) were incubated with the appropriate antibodies for 2 h at 4 uc, followed by the addition of 6 mg protein A-Sepharose (Sigma) and a further 2 h incubation at 4 uc. The beads were washed in 10 mm Tris/HCl ph 8.0, 1 mm EDTA, 1 mm PMSF and 0.1 % Igepal CA-630 containing 150 mm NaCl (one wash), 1 M NaCl (three washes) or 0.5 M LiCl (three washes). Complexes were eluted and cross-links reversed by the addition of 300 ml elution buffer (50 mm Tris/HCl, ph 6.8, 200 mm NaCl, 1 mm EDTA, 1 % SDS) and incubation at 65 uc for 15 h. Cell lysates (10 ml) mixed with 300 ml elution buffer were also incubated at 65 uc for 15 h to obtain an input RNA sample. Proteins were digested with proteinase K, nucleic acids were extracted with phenol/chloroform and precipitated with ethanol in the presence of trna carrier. Quantitative analysis of viral RNAs. Viral RNAs were quantified by primer extension assay or quantitative RT-qPCR. Primer extension analysis of viral NA, NP and M gene-specific RNAs and cellular 5S rrna were performed as described previously (Hara et al., 2006; Robb et al., 2009). For quantitative RT-qPCR, viral RNAs were reverse transcribed by using primers specific for the NP and M vrna and crna or a T 20 primer for mrna with SuperScript III reverse transcriptase (Invitrogen) at 50 uc for 1 h (Supplementary Table S1, available in JGV Online). Resulting cdnas were quantified by using sets of PCR primers specific for the NP and M vrna, crna and mrna (Supplementary Table S1) and the QuantiTect SYBR Green PCR kit (Qiagen) in a Corbett Rotor-Gene RG-3000 cycler. Reactions were set up in triplicate and data were analysed by using the Rotor- Gene 6 software. A standard curve was obtained for each set of PCR primers by analysing serial dilutions of ppoli-np-rt or ppoli-m- RT (Fodor et al., 1999). The amounts of immunoprecipitated RNA (Supplementary Table S2, available in JGV Online) were expressed as per cent of RNA present in cell lysates. ACKNOWLEDGEMENTS We thank J. Ortín, T. Toyoda and R. Reed for antibodies, P. Palese for viruses and F. Vreede, N. Robb and G. G. 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