The accumulation of influenza A virus segment 7 spliced mrnas is regulated by the NS1 protein

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1 Journal of General Virology (2012), 93, DOI /vir Short Communication Correspondence Ervin Fodor Received 24 June 2011 Accepted 12 September 2011 The accumulation of influenza A virus segment 7 spliced mrnas is regulated by the NS1 protein Nicole C. Robb and Ervin Fodor Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK The influenza A virus M1 mrna is alternatively spliced to produce M2 mrna, mrna 3, and in some cases, M4 mrna. Splicing of influenza mrnas is carried out by the cellular splicing machinery and is thought to be regulated, as both spliced and unspliced mrnas encode proteins. In this study, we used radioactively labelled primers to investigate the accumulation of spliced and unspliced M segment mrnas in viral infection and ribonucleoprotein (RNP) reconstitution assays in which only the minimal components required for transcription and replication to occur were expressed. We found that co-expression of the viral NS1 protein in an RNP reconstitution assay altered the accumulation of spliced mrnas compared with when it was absent, and that this activity was dependent on the RNA-binding ability of NS1. These findings suggest that the NS1 protein plays a role in the regulation of splicing of influenza virus M1 mrna. The genome of the influenza A virus consists of eight viral RNA (vrna) segments. Each vrna segment is bound by a single copy of the vrna polymerase complex consisting of the PA, PB1 and PB2 proteins, and multiple copies of the nucleoprotein (NP), to form a ribonucleoprotein (RNP) complex. The RNA polymerase is responsible for transcribing mrna from the vrna templates and replicating the vrna segments via complementary RNA (crna) intermediates (reviewed by Engelhardt & Fodor, 2006). The mrna species from segment 7 (M) and segment 8 (NS) undergo splicing to encode more than one protein. Segment 7 encodes the M1 matrix protein from unspliced M1 mrna, while alternative splicing gives rise to three spliced mrna products. The three spliced transcripts share a common 39 splice site but have alternative 59 splice sites (Fig. 1a). Use of the first 59 splice site located at position 11 of the M1 mrna (numbering refers to nucleotide positions in the vrna template) gives rise to mrna 3 (Lamb et al., 1981), which has the potential to encode an as yet undiscovered 9 aa peptide. Use of the next 59 splice site located at position 51 gives rise to M2 mrna, which encodes the M2 ion channel protein (Lamb & Choppin, 1981; Winter & Fields, 1980). The final 59 splice site, located at position 145, gives rise to M4 mrna, which could potentially encode a 54 aa protein with the first 40 aa identical to the N terminus of the M1 protein (Shih et al., 1998). Although mrna 3 and M2 mrna are found in all viral strains, M4 mrna only exists in some strains, including A/WSN/33. The 59 and 39 splice sites of influenza mrnas resemble the consensus sequences for the exon/intron boundaries of cellular mrna transcripts (Mount, 1982) and splicing of influenza mrna has been demonstrated in the absence of other viral proteins (Lamb & Lai, 1982), indicating that the virus is using the cellular splicing machinery. Splicing of the M1 mrna is thought to be regulated (Valcárcel et al., 1991), possibly by the cellular splicing factor SF2/ASF (Shih & Krug, 1996) and the viral polymerase proteins (Shih et al., 1995), although a recent study has challenged this (Bier et al., 2011). In addition, it was found that the viral NS1 protein inhibited the splicing of cellular transcripts and M1 mrna (Lu et al., 1994). In contrast, a second study found no differences in M1 and M2 protein levels between wildtype influenza virus and a recombinant virus lacking NS1, suggesting that the NS1 protein does not play a role in the regulation of M1 mrna splicing (Salvatore et al., 2002). To investigate the regulation of M1 mrna splicing, we used a quantitative primer extension assay (Robb et al., 2010) to analyse the accumulation of spliced and unspliced mrnas derived from the M segment (Fig. 1a). M segment RNAs were initially analysed using the 890 and 904 primers in 293T cells infected with A/WSN/33. The 904 primer was appropriate for the detection of M2, mrna 3 and M4 mrna; however, the primer extension products from the M1 mrna and M crna could not be resolved (Fig. 1b). The samples were therefore also analysed using the 162 primer, which produces a shorter M1 mrna-specific product that can be resolved from the M crna-specific band due to the presence of a 59 capped primer in the mrna (Fig. 1c). The mrnas were quantified using an FLA-5000 Fluoro Image Analyser (Fuji) and AIDA software (Raytest) (Fig. 1d) and their accumulation was found to remain at similar levels over the course of the infection. The majority of mrna detected was spliced, as unspliced M1 mrna represented only 8 % [on average between 4 and 10 h post-infection (p.i.)] of total M segment-specific G 2012 SGM Printed in Great Britain 113

2 N. C. Robb and E. Fodor Fig. 1. Splicing of M1 mrna during viral infection. (a) Diagram of the RNA species of the A/WSN/33 M segment and the design of primers. Squares at the 59 end of the mrnas represent the 59 cap and AAA at the 39 end represents the poly(a) tail. A forward primer (59-GAAAAGAGGGCCTTCTACGG-39) annealing at position 890 detects M vrna, a reverse primer annealing at position 162 (59-AGCCATTCCATGAGAACCTC-39) detects both M1 mrna and M crna, and a second reverse primer annealing at position 904 (59-GAAGGCCCTCTTTTCAAACC-39) detects M crna and all M mrnas. Expected sizes of cdna products are listed to the right of the figure, the 59 capped primer was estimated to be 13 nt. (b) Analysis of the accumulation of A/WSN/33 M segment mrnas during viral infection. 293T cells were infected with A/WSN/33 at an m.o.i. of 2.5 and RNA was harvested at regular times p.i. RNA species were analysed using the 890 and 904 primers. (c) RNA samples were also analysed using the 162 primer to resolve M1 mrna and M crna. (d) Quantification of spliced and unspliced mrnas expressed as a percentage of the total M segment mrnas, averaged from three independent experiments. Error bars represent SD. ND, Non-detectable. mrna. Spliced M2 mrna was 63 % of total mrna, while mrna 3 was 13 % and M4 mrna was 16 % (Fig. 1d). It has previously been suggested that M2 mrna represents only a small proportion of the total mrna (Lamb et al., 1981); however, in that study the A/Udorn/72 virus strain was used, suggesting the possibility that different viral strains exhibit different splicing efficiencies of M1 mrna. Next, the accumulation of M segment mrnas was evaluated in a viral RNP reconstitution assay involving only the minimal components required for viral transcription and replication (the RNA polymerase, the nucleoprotein and a vrna template). 293T cells were transfected with plasmids expressing the A/WSN/33 PB1, PB2, PA and NP proteins and an M vrna template; and total RNA was harvested and analysed by primer extension (Fig. 2a c). M1 mrna was 22 % [on average between 24 and 48 h post-transfection (p.t.)] of total M segment mrna, while M2 mrna was 38 %, mrna 3 was 35 % and M4 mrna was 5 % (Fig. 2c). The accumulation of M2 and M4 mrnas therefore decreased and the proportion of mrna 3 increased in an RNP reconstitution assay compared with infection. As only the minimal components required for transcription and replication were present in the transfection system this suggested 114 Journal of General Virology 93

Splicing of influenza virus M1 mrna Fig. 2. Splicing of M1 mrna in RNP reconstitution assays. (a) Analysis of the splicing of M1 mrna in the absence of NS1.

3 Splicing of influenza virus M1 mrna Fig. 2. Splicing of M1 mrna in RNP reconstitution assays. (a) Analysis of the splicing of M1 mrna in the absence of NS1. 293T cells were transfected with pcdna plasmids expressing the A/WSN/33 PB1, PB2, PA and NP proteins and ppoli-m-rt expressing the M vrna template (Fodor et al., 1999, 2002), and RNA was harvested at regular times p.t. M RNA species were analysed using the 890 and 904 primers. (b) RNA samples were also analysed using the 162 primer to resolve M1 mrna and M crna. (c) Quantification of the accumulation of spliced and unspliced mrnas expressed as a percentage of the total M segment mrnas, averaged from three independent experiments. Error bars represent SD. ND, Non-detectable. (d f) Analysis of splicing of M1 mrna in the presence of NS1. 293T cells were transfected with pcdna plasmids expressing the PB1, PB2, PA, NP and NS1 proteins (Robb et al., 2010) and ppoli-m-rt expressing M vrna. RNA species were analysed using a similar approach as described above. (g i) Analysis of splicing of M1 mrna in the presence of NS1 K38A/R41A (Robb et al., 2011). that another viral protein(s), which was not present, may play a role in the regulation of M1 mrna splicing. As the NS1 protein has previously been suggested as a candidate for splicing regulation (Lu et al., 1994), an RNP reconstitution assay in which NS1 was additionally expressed was carried out. 293T cells were transfected with plasmids expressing the PB1, PB2, PA and NP proteins, M vrna, and the NS1 protein. NS1 protein expression was confirmed by Western blot analysis (Fig. 2d) and RNA was 115

4 N. C. Robb and E. Fodor analysed by primer extension (Fig. 2d f). NS1 expression altered the accumulation of spliced mrnas by decreasing the accumulation of mrna 3 to 14 % of total M segment mrna, compared with 35 % in an RNP reconstitution assay where NS1 was absent (compare Fig. 2c to Fig. 2f). The proportions of M2 and M4 mrna were 37 and 7 % of total M segment mrna respectively, similar to those found when NS1 was not expressed. The N-terminal RNA-binding domain of NS1 has been shown to interact with a variety of double and singlestranded RNA species (Hatada & Fukuda, 1992; Hatada et al., 1992, 1997; Lu et al., 1995; Marión et al., 1997; Qiu & Krug, 1994; Qiu et al., 1995). Alanine mutations at residues 38 and 41 have previously been shown to abrogate the RNA-binding activity of NS1 (Wang et al., 1999). In order to test whether the RNA-binding ability of NS1 was required for altered M1 mrna splicing 293T cells were transfected with plasmids expressing the PB1, PB2, PA and NP proteins, M vrna, and mutant NS1 K38A/R41A protein. Expression of NS1 K38A/R41A was confirmed by Western blot and RNA species were analysed by primer extension (Fig. 2g i). Expression of NS1 K38A/R41A appears to be lower than that of wild-type NS1, although it is not clear whether the mutations in NS1 decreased protein expression or resulted in less sensitive antibody detection. The proportion of M1 mrna was 31 % of total M segment mrna, while M2 mrna was 39 %, mrna 3 was 26 % and M4 mrna was 4 % (Fig. 2i). Thus, the proportion of M1 mrna decreased, while mrna 3 increased compared with when wild-type NS1 was present (Fig. 2f), suggesting that the effect on the accumulation of spliced mrna by NS1 is dependent on the protein s ability to bind RNA. The vrna polymerase has previously been suggested to regulate the splicing of M1 mrna. It was proposed that early in infection mrna 3 is predominantly produced, until at later time points newly synthesized polymerase proteins bind to the 59 end of the mrna and block the mrna 3 splice site, allowing M2 mrna to be made (Shih et al., 1995). The effect of NS1 expression on viral polymerase accumulation in the RNP reconstitution assay was therefore investigated. 293T cells were transfected with plasmids expressing the PB1, PB2, PA and NP proteins, and either empty plasmid or plasmids expressing wild-type or K38A/ R41A NS1. Cell lysates were harvested after 0, 24 and 48 h, and analysed by Western blot. Expression of wild-type and K38A/R41A NS1 proteins was confirmed and the detection of b-actin served as a loading control (Fig. 3a). In the absence of NS1, expression of PB1, PB2, PA and NP peaked at 24 h p.t., whereas in the presence of wild-type or mutant 116 Journal of General Virology 93 Fig. 3. The polymerase complex is unlikely to regulate M1 mrna splicing. (a) 293T cells were transfected with pcdna plasmids expressing the PB1, PB2, PA and NP proteins, and either empty plasmid or plasmids expressing wild-type or K38A/R41A NS1. Cell lysates were harvested after 0, 24 and 48 h, and analysed by Western blot using antibodies against the viral PB1, PB2, PA, NP, NS1 and cellular b-actin proteins. (b) 293T cells were transfected with pcdna plasmids expressing the PB1, PB2, PA, NP and NS1 proteins (with PA and NS1 either TAP-tagged or untagged) (Robb et al., 2011), and ppoli-m-rt expressing M vrna. Cell lysates were harvested after 24 h and purified by IgG Sepharose chromatography using the TAP-tag (as described in Robb et al., 2011). Proteins were analysed by Western blot and RNA by primer extension (Bier et al., 2011). *Note that TAP-tagged NS1 and PA proteins run more slowly than the purified products cleaved with TEV protease.

5 Splicing of influenza virus M1 mrna NS1 the accumulation of these proteins was delayed (Fig. 3a). NS1 has previously been implicated in the inhibition of 39 end processing and nuclear export of mrnas transcribed by cellular RNA polymerase II (Chen et al., 1999; Nemeroff et al., 1998), possibly explaining the lower accumulation of polymerase proteins and NP from plasmids in the RNP reconstitution assay. Overall, there was no correlation between polymerase expression and the level of mrna 3 observed in RNP reconstitution assays. In fact, the decreased mrna 3 accumulation observed when wild-type NS1 is expressed in an RNP reconstitution assay (Fig. 2d f) occurs despite the less rapid accumulation of polymerase proteins. These results suggest that the effect of NS1 expression on M1 mrna splicing is unlikely to be linked to regulation by the polymerase complex. In addition, in an RNP reconstitution experiment where TAP-tagged NS1 or PA proteins were purified and the associated RNA species were analysed by primer extension, it was found that M1 mrna co-purified with NS1, while no detectable M1 mrna was associated with the PA protein (Fig. 3b). The binding of NS1 to M1 mrna and the absence of a stable interaction between the vrna polymerase and viral M1 mrna further suggest that the regulation of splicing may be carried out by the NS1 protein and not the polymerase complex. Unlike cellular mrnas, which are fully spliced so that usually only the spliced mrna transcripts are found in the cytoplasm, influenza virus mrna splicing is regulated to allow expression of proteins from both spliced and unspliced transcripts. In this study, we have shown that expression of NS1 altered the accumulation of spliced mrnas compared with when it was absent, suggesting that NS1 plays a role in the regulation of splicing of influenza virus M1 mrna. The primary activity of NS1 during viral infection is the suppression of host immune responses, although it has also been implicated in many other processes (reviewed by Hale et al., 2008). NS1 has been shown to inhibit the nuclear export of cellular mrnas (Chen & Krug, 2000; Fortes et al., 1994; Qiu & Krug, 1994; Satterly et al., 2007), and there is increasing evidence to suggest that it may also be involved in the export of viral mrnas (Alonso-Caplen & Krug, 1991; Garaigorta & Ortín, 2007; Schneider & Wolff, 2009; Wang et al., 2008). It is therefore conceivable that NS1 affects the accumulation of M mrnas by influencing the rate of transport of unspliced and/or spliced mrnas. As the RNAbinding ability of NS1 appears to be required for this activity it is possible that NS1 is acting as an adaptor protein between viral mrnas and cellular export factors. Alternatively, NS1 may be acting by directly recruiting cellular splicing factors to the viral mrna. An example may be SF2/ASF, a member of the SR family of proteins that are essential for the formation of the spliceosome, and has been shown to affect 59 splice site selection in a concentrationdependent manner (Ge & Manley, 1990). Mutations inhibiting the interaction of NS1 with the cellular factors PKR and CPSF-30 had no effect on the ability of NS1 to regulate M1 mrna splicing (data not shown). Additional studies are required to understand the mechanism of splicing regulation by NS1. Acknowledgements We thank Adolfo García-Sastre and Otto Haller for antibodies. This work was supported by the MRC (G ). References Alonso-Caplen, F. V. & Krug, R. M. (1991). Regulation of the extent of splicing of influenza virus NS1 mrna: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mrna. Mol Cell Biol 11, Bier, K., York, A. & Fodor, E. (2011). Cellular cap-binding proteins associate with influenza virus mrnas. J Gen Virol 92, Chen, Z. & Krug, R. M. (2000). 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6 N. C. Robb and E. Fodor Lamb, R. A., Lai, C. J. & Choppin, P. W. (1981). Sequences of mrnas derived from genome RNA segment 7 of influenza virus: colinear and interrupted mrnas code for overlapping proteins. Proc Natl Acad Sci USA78, Lu, Y., Qian, X. Y. & Krug, R. M. (1994). The influenza virus NS1 protein: a novel inhibitor of pre-mrna splicing. Genes Dev 8, Lu, Y., Wambach, M., Katze, M. G. & Krug, R. M. (1995). Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor. Virology 214, Marión, R. M., Aragón, T., Beloso, A., Nieto, A. & Ortín, J. (1997). The N-terminal half of the influenza virus NS1 protein is sufficient for nuclear retention of mrna and enhancement of viral mrna translation. Nucleic Acids Res 25, Mount, S. M. (1982). A catalogue of splice junction sequences. Nucleic Acids Res 10, Nemeroff, M. E., Barabino, S. M., Li, Y., Keller, W. & Krug, R. M. (1998). Influenza virus NS1 protein interacts with the cellular 30 kda subunit of CPSF and inhibits 3 end formation of cellular pre-mrnas. Mol Cell 1, Qiu, Y. & Krug, R. M. (1994). The influenza virus NS1 protein is a poly(a)-binding protein that inhibits nuclear export of mrnas containing poly(a). J Virol 68, Qiu, Y., Nemeroff, M. & Krug, R. M. (1995). The influenza virus NS1 protein binds to a specific region in human U6 snrna and inhibits U6-U2 and U6-U4 snrna interactions during splicing. RNA 1, Robb, N. C., Jackson, D., Vreede, F. T. & Fodor, E. (2010). Splicing of influenza A virus NS1 mrna is independent of the viral NS1 protein. J Gen Virol 91, Robb, N. C., Chase, G., Bier, K., Vreede, F. T., Shaw, P. C., Naffakh, N., Schwemmle, M. & Fodor, E. (2011). The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes. J Virol 85, Salvatore, M., Basler, C. F., Parisien, J. P., Horvath, C. M., Bourmakina, S., Zheng, H., Muster, T., Palese, P. & García-Sastre, A. (2002). Effects of influenza A virus NS1 protein on protein expression: the NS1 protein enhances translation and is not required for shutoff of host protein synthesis. JVirol76, Satterly, N., Tsai, P. L., van Deursen, J., Nussenzveig, D. R., Wang, Y., Faria, P. A., Levay, A., Levy, D. E. & Fontoura, B. M. (2007). Influenza virus targets the mrna export machinery and the nuclear pore complex. Proc Natl Acad Sci U S A 104, Schneider, J. & Wolff, T. (2009). Nuclear functions of the influenza A and B viruses NS1 proteins: do they play a role in viral mrna export? Vaccine 27, Shih, S. R. & Krug, R. M. (1996). Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells. EMBO J 15, Shih, S. R., Nemeroff, M. E. & Krug, R. M. (1995). The choice of alternative 59 splice sites in influenza virus M1 mrna is regulated by the viral polymerase complex. Proc Natl Acad Sci U S A 92, Shih, S. R., Suen, P. C., Chen, Y. S. & Chang, S. C. (1998). Anovel spliced transcript of influenza A/WSN/33 virus. Virus Genes 17, Valcárcel, J., Portela, A. & Ortín, J. (1991). Regulated M1 mrna splicing in influenza virus-infected cells. J Gen Virol 72, Wang, W., Riedel, K., Lynch, P., Chien, C. Y., Montelione, G. T. & Krug, R. M. (1999). RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. RNA 5, Wang, W., Cui, Z. Q., Han, H., Zhang, Z. P., Wei, H. P., Zhou, Y. F., Chen, Z. & Zhang, X. E. (2008). Imaging and characterizing influenza A virus mrna transport in living cells. Nucleic Acids Res 36, Winter, G. & Fields, S. (1980). Cloning of influenza cdna into M13: the sequence of the RNA segment encoding the A/PR/8/34 matrix protein. Nucleic Acids Res 8, Journal of General Virology 93

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