1887

Abstract

The influenza virus RNA polymerase transcribes the negative-sense viral RNA segments (vRNA) into mRNA and replicates them via complementary RNA (cRNA) intermediates into more copies of vRNA. It is not clear how the relative amounts of the three RNA products, mRNA, cRNA and vRNA, are regulated during the viral life cycle. We found that in viral ribonucleoprotein (vRNP) reconstitution assays involving only the minimal components required for viral transcription and replication (the RNA polymerase, the nucleoprotein and a vRNA template), the relative levels of accumulation of RNA products differed from those observed in infected cells, suggesting a regulatory role for additional viral proteins. Expression of the viral NS2/NEP protein in RNP reconstitution assays affected viral RNA levels by reducing the accumulation of transcription products and increasing the accumulation of replication products to more closely resemble those found during viral infection. This effect was functionally conserved in influenza A and B viruses and was influenza-virus-type-specific, demonstrating that the NS2/NEP protein changes RNA levels by specific alteration of the viral transcription and replication machinery, rather than through an indirect effect on the host cell. Although NS2/NEP has been shown previously to play a role in the nucleocytoplasmic export of viral RNPs, deletion of the nuclear export sequence region that is required for its transport function did not affect the ability of the protein to regulate RNA levels. A role for the NS2/NEP protein in the regulation of influenza virus transcription and replication that is independent of its viral RNP export function is proposed.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.009639-0
2009-06-01
2024-11-05
Loading full text...

Full text loading...

/deliver/fulltext/jgv/90/6/jgv001398.html?itemId=/content/journal/jgv/10.1099/vir.0.009639-0&mimeType=html&fmt=ahah

References

  1. 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]
  2. Baudin F., Petit I., Weissenhorn W., Ruigrok R. W. 2001; In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology 281:102–108 [CrossRef]
    [Google Scholar]
  3. Braam J., Ulmanen I., Krug R. M. 1983; Molecular model of a eucaryotic transcription complex: functions and movements of influenza P proteins during capped RNA-primed transcription. Cell 34:609–618
    [Google Scholar]
  4. Bullido R., Gómez-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]
  5. Deng T., Vreede F. T., Brownlee G. G. 2006; Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication. J Virol 80:2337–2348 [CrossRef]
    [Google Scholar]
  6. Elster C., Larsen K., Gagnon J., Ruigrok R. W., Baudin F. 1997; Influenza virus M1 protein binds to RNA through its nuclear localization signal. J Gen Virol 78:1589–1596
    [Google Scholar]
  7. Elton D., Simpson-Holley M., Archer K., Medcalf L., Hallam R., McCauley J., Digard P. 2001; Interaction of the influenza virus nucleoprotein with the cellular CRM1-mediated nuclear export pathway. J Virol 75:408–419 [CrossRef]
    [Google Scholar]
  8. 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]
  9. Fodor E., Devenish L., Engelhardt O. G., Palese P., Brownlee G. G., García-Sastre A. 1999; Rescue of influenza A virus from recombinant DNA. J Virol 73:9679–9682
    [Google Scholar]
  10. 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]
  11. Gabriel G., Dauber B., Wolff T., Planz O., Klenk H. D., Stech J. 2005; The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci U S A 102:18590–18595 [CrossRef]
    [Google Scholar]
  12. Imai M., Watanabe S., Odagiri T. 2003; Influenza B virus NS2, a nuclear export protein, directly associates with the viral ribonucleoprotein complex. Arch Virol 148:1873–1884 [CrossRef]
    [Google Scholar]
  13. Inglis S. C., Barrett T., Brown C. M., Almond J. W. 1979; The smallest genome RNA segment of influenza virus contains two genes that may overlap. Proc Natl Acad Sci U S A 76:3790–3794 [CrossRef]
    [Google Scholar]
  14. Jackson D., Cadman A., Zurcher T., Barclay W. S. 2002; A reverse genetics approach for recovery of recombinant influenza B viruses entirely from cDNA. J Virol 76:11744–11747 [CrossRef]
    [Google Scholar]
  15. Kohno Y., Muraki Y., Matsuzaki Y., Takashita E., Sugawara K., Hongo S. 2009; Intracellular localization of influenza C virus NS2 protein (NEP) in infected cells and its incorporation into virions. Arch Virol 154:235–243 [CrossRef]
    [Google Scholar]
  16. Krug R. M. 1981; Priming of influenza viral RNA transcription by capped heterologous RNAs. Curr Top Microbiol Immunol 93:125–149
    [Google Scholar]
  17. Labadie K., Dos Santos Afonso E., Rameix-Welti M. A., van der Werf S., Naffakh N. 2007; Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells. Virology 362:271–282 [CrossRef]
    [Google Scholar]
  18. Lamb R. A., Choppin P. W. 1979; Segment 8 of the influenza virus genome is unique in coding for two polypeptides. Proc Natl Acad Sci U S A 76:4908–4912 [CrossRef]
    [Google Scholar]
  19. Lamb R. A., Lai C. J. 1980; Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus. Cell 21:475–485 [CrossRef]
    [Google Scholar]
  20. Lee K. H., Seong B. L. 1998; The position 4 nucleotide at the 3′ end of the influenza virus neuraminidase vRNA is involved in temporal regulation of transcription and replication of neuraminidase RNAs and affects the repertoire of influenza virus surface antigens. J Gen Virol 79:1923–1934
    [Google Scholar]
  21. Lee M. K., Bae S. H., Park C. J., Cheong H. K., Cheong C., Choi B. S. 2003; A single-nucleotide natural variation (U4 to C4) in an influenza A virus promoter exhibits a large structural change: implications for differential viral RNA synthesis by RNA-dependent RNA polymerase. Nucleic Acids Res 31:1216–1223 [CrossRef]
    [Google Scholar]
  22. Mullin A. E., Dalton R. M., Amorim M. J., Elton D., Digard P. 2004; Increased amounts of the influenza virus nucleoprotein do not promote higher levels of viral genome replication. J Gen Virol 85:3689–3698 [CrossRef]
    [Google Scholar]
  23. Neumann G., Hughes M. T., Kawaoka Y. 2000; Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1. EMBO J 19:6751–6758 [CrossRef]
    [Google Scholar]
  24. Odagiri T., Tobita K. 1990; Mutation in NS2, a nonstructural protein of influenza A virus, extragenically causes aberrant replication and expression of the PA gene and leads to generation of defective interfering particles. Proc Natl Acad Sci U S A 87:5988–5992 [CrossRef]
    [Google Scholar]
  25. Odagiri T., Tominaga K., Tobita K., Ohta S. 1994; An amino acid change in the non-structural NS2 protein of an influenza A virus mutant is responsible for the generation of defective interfering (DI) particles by amplifying DI RNAs and suppressing complementary RNA synthesis. J Gen Virol 75:43–53 [CrossRef]
    [Google Scholar]
  26. 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]
  27. Palese P. 1977; The genes of influenza virus. Cell 10:1–10 [CrossRef]
    [Google Scholar]
  28. Paragas J., Talon J., O'Neill R. E., Anderson D. K., García-Sastre A., Palese P. 2001; Influenza B and C virus NEP (NS2) proteins possess nuclear export activities. J Virol 75:7375–7383 [CrossRef]
    [Google Scholar]
  29. Pleschka S., Jaskunas R., Engelhardt O. G., Zurcher T., Palese P., García-Sastre A. 1996; A plasmid-based reverse genetics system for influenza A virus. J Virol 70:4188–4192
    [Google Scholar]
  30. Portela A., Digard P. 2002; The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83:723–734
    [Google Scholar]
  31. Richardson J. C., Akkina R. K. 1991; NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells. Arch Virol 116:69–80 [CrossRef]
    [Google Scholar]
  32. Salomon R., Franks J., Govorkova E. A., Ilyushina N. A., Yen H. L., Hulse-Post D. J., Humberd J., Trichet M., Rehg J. E. other authors 2006; The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04. J Exp Med 203:689–697 [CrossRef]
    [Google Scholar]
  33. 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]
  34. Watanabe K., Handa H., Mizumoto K., Nagata K. 1996; Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein. J Virol 70:241–247
    [Google Scholar]
  35. Yasuda J., Nakada S., Kato A., Toyoda T., Ishihama A. 1993; Molecular assembly of influenza virus: association of the NS2 protein with virion matrix. Virology 196:249–255 [CrossRef]
    [Google Scholar]
  36. Ye Z. P., Pal R., Fox J. W., Wagner R. R. 1987; Functional and antigenic domains of the matrix (M1) protein of influenza A virus. J Virol 61:239–246
    [Google Scholar]
  37. Ye Z. P., Baylor N. W., Wagner R. R. 1989; Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides. J Virol 63:3586–3594
    [Google Scholar]
/content/journal/jgv/10.1099/vir.0.009639-0
Loading
/content/journal/jgv/10.1099/vir.0.009639-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
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