1887

Abstract

RNA segment 8 (NS) of influenza A virus encodes two proteins. The NS1 protein is translated from the unspliced primary mRNA transcript, whereas the second protein encoded by this segment, NS2/NEP, is translated from a spliced mRNA. Splicing of influenza NS1 mRNA is thought to be regulated so that the levels of NS2 spliced transcripts are approximately 10 % of total NS mRNA. Regulation of splicing of the NS1 mRNA has been studied at length, and a number of often-contradictory control mechanisms have been proposed. In this study, we used P-labelled gene-specific primers to investigate influenza A NS1 mRNA splicing regulation. It was found that the efficiency of splicing of NS1 mRNA was maintained at similar levels in both virus infection and ribonucleoprotein-reconstitution assays, and NS2 mRNA comprised approximately 15 % of total NS mRNA in both assays. The effect of NS1 protein expression on the accumulation of viral NS2 mRNA and spliced cellular -globin mRNA was analysed, and it was found that NS1 protein expression reduced spliced -globin mRNA levels, but had no effect on the accumulation of NS2 mRNA. We conclude that the NS1 protein specifically inhibits the accumulation of cellular RNA polymerase II-driven mRNAs, but does not affect the splicing of its own viral NS1 mRNA.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.022004-0
2010-09-01
2019-11-18
Loading full text...

Full text loading...

/deliver/fulltext/jgv/91/9/2331.html?itemId=/content/journal/jgv/10.1099/vir.0.022004-0&mimeType=html&fmt=ahah

References

  1. Agris, C. H., Nemeroff, M. E. & Krug, R. M. ( 1989; ). A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins. Mol Cell Biol 9, 259–267.
    [Google Scholar]
  2. Akarsu, H., Burmeister, W. P., Petosa, C., Petit, I., Muller, C. W., Ruigrok, R. W. & Baudin, F. ( 2003; ). Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). EMBO J 22, 4646–4655.[CrossRef]
    [Google Scholar]
  3. 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, 1092–1098.
    [Google Scholar]
  4. Alonso-Caplen, F. V., Nemeroff, M. E., Qiu, Y. & Krug, R. M. ( 1992; ). Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA. Genes Dev 6, 255–267.[CrossRef]
    [Google Scholar]
  5. Bullido, R., Gomez-Puertas, P., Saiz, M. J. & Portela, A. ( 2001; ). Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs. J Virol 75, 4912–4917.[CrossRef]
    [Google Scholar]
  6. Chen, W., Calvo, P. A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., O'Neill, R., Schickli, J. & other authors ( 2001; ). A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 7, 1306–1312.[CrossRef]
    [Google Scholar]
  7. Das, K., Ma, L. C., Xiao, R., Radvansky, B., Aramini, J., Zhao, L., Marklund, J., Kuo, R. L., Twu, K. Y. & other authors ( 2008; ). Structural basis for suppression of a host antiviral response by influenza A virus. Proc Natl Acad Sci U S A 105, 13093–13098.[CrossRef]
    [Google Scholar]
  8. Dias, A., Bouvier, D., Crepin, T., McCarthy, A. A., Hart, D. J., Baudin, F., Cusack, S. & Ruigrok, R. W. ( 2009; ). The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458, 914–918.[CrossRef]
    [Google Scholar]
  9. Engelhardt, O. G. & Fodor, E. ( 2006; ). Functional association between viral and cellular transcription during influenza virus infection. Rev Med Virol 16, 329–345.[CrossRef]
    [Google Scholar]
  10. Engelhardt, O. G., Smith, M. & Fodor, E. ( 2005; ). Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II. J Virol 79, 5812–5818.[CrossRef]
    [Google Scholar]
  11. Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G. & Garcia-Sastre, A. ( 1999; ). Rescue of influenza A virus from recombinant DNA. J Virol 73, 9679–9682.
    [Google Scholar]
  12. Fodor, E., Crow, M., Mingay, L. J., Deng, T., Sharps, J., Fechter, P. & Brownlee, G. G. ( 2002; ). A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J Virol 76, 8989–9001.[CrossRef]
    [Google Scholar]
  13. Fortes, P., Beloso, A. & Ortin, J. ( 1994; ). Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J 13, 704–712.
    [Google Scholar]
  14. Garaigorta, U. & Ortin, J. ( 2007; ). Mutation analysis of a recombinant NS replicon shows that influenza virus NS1 protein blocks the splicing and nucleo-cytoplasmic transport of its own viral mRNA. Nucleic Acids Res 35, 4573–4582.[CrossRef]
    [Google Scholar]
  15. Gultyaev, A. P. & Olsthoorn, R. C. ( 2010; ). A family of non-classical pseudoknots in influenza A and B viruses. RNA Biol 7, 125–129.[CrossRef]
    [Google Scholar]
  16. Gultyaev, A. P., Heus, H. A. & Olsthoorn, R. C. ( 2007; ). An RNA conformational shift in recent H5N1 influenza A viruses. Bioinformatics 23, 272–276.[CrossRef]
    [Google Scholar]
  17. Hale, B. G., Randall, R. E., Ortin, J. & Jackson, D. ( 2008; ). The multifunctional NS1 protein of influenza A viruses. J Gen Virol 89, 2359–2376.[CrossRef]
    [Google Scholar]
  18. Hirose, Y. & Manley, J. L. ( 2000; ). RNA polymerase II and the integration of nuclear events. Genes Dev 14, 1415–1429.
    [Google Scholar]
  19. Howe, K. J. ( 2002; ). RNA polymerase II conducts a symphony of pre-mRNA processing activities. Biochim Biophys Acta 1577, 308–324.[CrossRef]
    [Google Scholar]
  20. Inglis, S. C. & Brown, C. M. ( 1984; ). Differences in the control of virus mRNA splicing during permissive or abortive infection with influenza A (fowl plague) virus. J Gen Virol 65, 153–164.[CrossRef]
    [Google Scholar]
  21. Krug, R. M. ( 1981; ). Priming of influenza viral RNA transcription by capped heterologous RNAs. Curr Top Microbiol Immunol 93, 125–149.
    [Google Scholar]
  22. Kuo, R. L. & Krug, R. M. ( 2009; ). Influenza A virus polymerase is an integral component of the CPSF30–NS1A protein complex in infected cells. J Virol 83, 1611–1616.[CrossRef]
    [Google Scholar]
  23. Lamb, R. A. & Lai, C. J. ( 1982; ). Spliced and unspliced messenger RNAs synthesized from cloned influenza virus M DNA in an SV40 vector: expression of the influenza virus membrane protein (M1). Virology 123, 237–256.[CrossRef]
    [Google Scholar]
  24. Lamb, R. A. & Lai, C. J. ( 1984; ). Expression of unspliced NS1 mRNA, spliced NS2 mRNA, and a spliced chimera mRNA from cloned influenza virus NS DNA in an SV40 vector. Virology 135, 139–147.[CrossRef]
    [Google Scholar]
  25. Lamb, R. A., Etkind, P. R. & Choppin, P. W. ( 1978; ). Evidence for a ninth influenza viral polypeptide. Virology 91, 60–78.[CrossRef]
    [Google Scholar]
  26. Lamb, R. A., Choppin, P. W., Chanock, R. M. & Lai, C. J. ( 1980; ). Mapping of the two overlapping genes for polypeptides NS1 and NS2 on RNA segment 8 of influenza virus genome. Proc Natl Acad Sci U S A 77, 1857–1861.[CrossRef]
    [Google Scholar]
  27. 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 U S A 78, 4170–4174.[CrossRef]
    [Google Scholar]
  28. Li, Y., Chen, Z. Y., Wang, W., Baker, C. C. & Krug, R. M. ( 2001; ). The 3′-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo. RNA 7, 920–931.[CrossRef]
    [Google Scholar]
  29. Lu, Y., Qian, X. Y. & Krug, R. M. ( 1994; ). The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing. Genes Dev 8, 1817–1828.[CrossRef]
    [Google Scholar]
  30. Marion, R. M., Zurcher, T., de la Luna, S. & Ortin, J. ( 1997; ). Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo. J Gen Virol 78, 2447–2451.
    [Google Scholar]
  31. Min, J. Y., Li, S., Sen, G. C. & Krug, R. M. ( 2007; ). A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology 363, 236–243.[CrossRef]
    [Google Scholar]
  32. Nemeroff, M. E., Utans, U., Kramer, A. & Krug, R. M. ( 1992; ). Identification of cis-acting intron and exon regions in influenza virus NS1 mRNA that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes. Mol Cell Biol 12, 962–970.
    [Google Scholar]
  33. 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, 991–1000.[CrossRef]
    [Google Scholar]
  34. Odagiri, T., Tobita, K. & Tashiro, M. ( 1991; ). Synthesis of the NS 2 nonstructural protein messenger RNA of influenza A viruses occurs in the absence of viral protein synthesis. Arch Virol 120, 281–288.[CrossRef]
    [Google Scholar]
  35. O'Neill, R. E., Talon, J. & Palese, P. ( 1998; ). The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17, 288–296.[CrossRef]
    [Google Scholar]
  36. Plotch, S. J. & Krug, R. M. ( 1986; ). In vitro splicing of influenza viral NS1 mRNA and NS1–β-globin chimeras: possible mechanisms for the control of viral mRNA splicing. Proc Natl Acad Sci U S A 83, 5444–5448.[CrossRef]
    [Google Scholar]
  37. Poon, L. L., Pritlove, D. C., Fodor, E. & Brownlee, G. G. ( 1999; ). Direct evidence that the poly(A) tail of influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA template. J Virol 73, 3473–3476.
    [Google Scholar]
  38. Pritlove, D. C., Poon, L. L., Fodor, E., Sharps, J. & Brownlee, G. G. ( 1998; ). Polyadenylation of influenza virus mRNA transcribed in vitro from model virion RNA templates: requirement for 5′ conserved sequences. J Virol 72, 1280–1286.
    [Google Scholar]
  39. Proudfoot, N. J., Furger, A. & Dye, M. J. ( 2002; ). Integrating mRNA processing with transcription. Cell 108, 501–512.[CrossRef]
    [Google Scholar]
  40. 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, 2425–2432.
    [Google Scholar]
  41. 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, 304–316.
    [Google Scholar]
  42. Robb, N. C., Smith, M., Vreede, F. T. & Fodor, E. ( 2009; ). NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome. J Gen Virol 90, 1398–1407.[CrossRef]
    [Google Scholar]
  43. Rozen, S. & Skaletsky, H. ( 2000; ). Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132, 365–386.
    [Google Scholar]
  44. Shih, S. R., Suen, P. C., Chen, Y. S. & Chang, S. C. ( 1998; ). A novel spliced transcript of influenza A/WSN/33 virus. Virus Genes 17, 179–183.[CrossRef]
    [Google Scholar]
  45. Shimizu, K., Iguchi, A., Gomyou, R. & Ono, Y. ( 1999; ). Influenza virus inhibits cleavage of the HSP70 pre-mRNAs at the polyadenylation site. Virology 254, 213–219.[CrossRef]
    [Google Scholar]
  46. Skehel, J. J. ( 1972; ). Polypeptide synthesis in influenza virus-infected cells. Virology 49, 23–36.[CrossRef]
    [Google Scholar]
  47. Smith, D. B. & Inglis, S. C. ( 1985; ). Regulated production of an influenza virus spliced mRNA mediated by virus-specific products. EMBO J 4, 2313–2319.
    [Google Scholar]
  48. Valcarcel, J., Portela, A. & Ortin, J. ( 1991; ). Regulated M1 mRNA splicing in influenza virus-infected cells. J Gen Virol 72, 1301–1308.[CrossRef]
    [Google Scholar]
  49. Vreede, F. T., Jung, T. E. & Brownlee, G. G. ( 2004; ). Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates. J Virol 78, 9568–9572.[CrossRef]
    [Google Scholar]
  50. Wang, W. & Krug, R. M. ( 1998; ). U6atac snRNA, the highly divergent counterpart of U6 snRNA, is the specific target that mediates inhibition of AT–AC splicing by the influenza virus NS1 protein. RNA 4, 55–64.
    [Google Scholar]
  51. Wise, H. M., Foeglein, A., Sun, J., Dalton, R. M., Patel, S., Howard, W., Anderson, E. C., Barclay, W. S. & Digard, P. ( 2009; ). A complicated message: identification of a novel PB1-related protein translated from influenza A virus segment 2 mRNA. J Virol 83, 8021–8031.[CrossRef]
    [Google Scholar]
  52. Yuan, P., Bartlam, M., Lou, Z., Chen, S., Zhou, J., He, X., Lv, Z., Ge, R., Li, X. & other authors ( 2009; ). Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature 458, 909–913.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.022004-0
Loading
/content/journal/jgv/10.1099/vir.0.022004-0
Loading

Data & Media loading...

Supplements

vol. , part 9, pp. 2331–2340

Detection of spliced NS2 mRNA by primer extension is in the linear range.

The ratio of A/Udorn/72 unspliced NS1 mRNA and spliced NS2 mRNA remains constant during viral infection.

The ratio of A/WSN/33 unspliced NS1 mRNA and spliced NS2 mRNA remains constant during viral infection in DF-1 cells.

Splicing of the NS1 mRNA occurs in cycloheximide-treated cells.

[ Single PDF file] (162 KB)



PDF

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error