1887

Abstract

It has previously been demonstrated in this laboratory that an influenza virus-like chloramphenicol acetyltransferase (CAT) RNA could be expressed in COS-1 cells that synthesized all ten influenza A virus-encoded proteins from recombinant plasmids. It was also shown that supernatant fluids harvested from these cultures contained virus-like particles (VLPs) that could deliver an enclosed CAT RNA to MDCK cells. Here, it is shown that the levels of expression of the reporter gene in the COS-1 and/or MDCK cells can be altered drastically by modifying the concentrations of the recombinant plasmids transfected in the COS-1 cells. Thus, it was observed that overexpression of NS2 reduced CAT expression in COS-1 cells, whereas overexpression of M2 and NS1 proteins dramatically decreased transmission of the CAT RNA to the MDCK cultures. These results are discussed with reference to the roles of these proteins during virus replication. From these experiments, a ratio of transfected plasmids was found that increased the efficiency of the previously described system by 50-100-fold. Under these optimized conditions, it was demonstrated that VLPs can be formed in the absence of neuraminidase expression and that these VLPs remained aggregated to each other and to cell membranes. Moreover, it is shown that CAT RNA transmission was dependent on specific interactions of the ribonucleoprotein complex with other viral structural polypeptides. These data demonstrate the usefulness of this encapsidation-packaging system for the study of different aspects of the influenza virus life-cycle.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-80-7-1635
1999-07-01
2022-10-03
Loading full text...

Full text loading...

/deliver/fulltext/jgv/80/7/0801635a.html?itemId=/content/journal/jgv/10.1099/0022-1317-80-7-1635&mimeType=html&fmt=ahah

References

  1. Alonso-Caplen F. V., Krug R. M. 1991; Regulat ion of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and ofthe nucleocytoplasmic transport of NS1 mRNA. Molecular and Cellular Biology 11:1092–1098
    [Google Scholar]
  2. Arrese M., Portela A. 1996; Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75. Journal of Virology 70:3385–3391
    [Google Scholar]
  3. Barclay W. S., Palese P. 1995; Influenza B viruses with site-specific mutations introduced into the HA gene. Journal of Virology 69:1275–1279
    [Google Scholar]
  4. Bridgen A., Elliott R. M. 1996; Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proceedings of the National Academy of Sciences, USA 93:15400–15404
    [Google Scholar]
  5. Compans R. W., Dimmock N. J. 1969; An electron microscopic study of single-cycle infection of chick embryo fibroblasts by influenza virus. Virology 39:499–515
    [Google Scholar]
  6. Conzelmann K. K., Schnell M. 1994; Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins. Journal of Virology 68:713–719
    [Google Scholar]
  7. Dunn E. F., Pritlove D. C., Jin H., Elliott R. M. 1995; Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins. Virology 211:133–143
    [Google Scholar]
  8. Enami K., Sato T. A., Nakada S., Enami M. 1994; Influenza virus NS1 protein stimulates translation of the M1 protein. Journal of Virology 68:1432–1437
    [Google Scholar]
  9. Fuerst T. R., Niles E. G., Studier F. W., Moss B. 1986; Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proceedings ofthe National Academy of Sciences, USA 83:8122–8126
    [Google Scholar]
  10. García-Sastre A., Palese P. 1995; The cytoplasmic tail of the neuraminidase protein of influenza A virus does not play an important role in the packaging of this protein into viral envelopes. Virus Research 37:37–47
    [Google Scholar]
  11. Henkel J. R., Weisz O. A. 1998; Influenza virus M2 protein slows traffic along the secretory pathway. pH perturbation of acidified compartments affects early Golgi transport steps. Journal of Biological Chemistry 273:6518–6524
    [Google Scholar]
  12. Herz C., Stavnezer E., Krug R. M., Gurney T. Jr 1981; Influenza virus, an RNA virus, synthesizes its messenger RNA in the nucleus of infected cells. Cell 26:391–400
    [Google Scholar]
  13. Huang T.-S., Palese P., Krystal M. 1990; Determination of influenza virus proteins required for genome replication. Journal of Virology 64:5669–5673
    [Google Scholar]
  14. Jambrina E., Barcena J., Uez O., Portela A. 1997; The three subunits of the polymerase and the nucleoprotein of influenza B virus are the minimum set of viral proteins required for expression of a model RNA template. Virology 235:209–217
    [Google Scholar]
  15. Jin H., Leser G. P., Lamb R. A. 1994; The influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity. EMBO Journal 13:5504–5515
    [Google Scholar]
  16. Jin H., Leser G. P., Zhang J., Lamb R. A. 1997; Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape. EMBO Journal 16:1236–1247
    [Google Scholar]
  17. Klumpp K., Ruigrok R. W. H., Baudin F. 1997; Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. EMBO Journal 16:1248–1257
    [Google Scholar]
  18. Lamb R. A. 1989; Genes and proteins of the influenza viruses. In The Influenza Viruses pp 1–87 Edited by Krug R. M. New York: Plenum;
    [Google Scholar]
  19. Lamb R. A., Krug R. M. 1996; Orthomyxoviridae: the viruses and their replication. In Fields Virology 3rd edn, pp 1353–1395 Edited by Fields B. N. Knipe D. M., Howley P. M. Philadelphia: Lippincott-Raven;
    [Google Scholar]
  20. 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
    [Google Scholar]
  21. 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. Proceedings of the National Academy of Sciences, USA 78:4170–4174
    [Google Scholar]
  22. Liu C., Eichelberger M. C., Compans R. W., Air G. M. 1995; Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding. Journal of Virology 69:1099–1106
    [Google Scholar]
  23. Lopez J. A., Guillen M., Sanchez-Fauquier A., Melero J. A. 1986; An antigen-binding assay to determine the specificity of monoclonal antibodies against influenza virus and mapping of epitopes. Journal of Virological Methods 13:255–264
    [Google Scholar]
  24. Marion R. M., Zurcher T., de la Luna S., Ortln J. 1997; Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo . Journal of General Virology 78:2447–2451
    [Google Scholar]
  25. Martin K., Helenius A. 1991; Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67:117–130
    [Google Scholar]
  26. Mena I., de la Luna S., Albo C., Martin J., Nieto A., Orti’n J., Portela A. 1994; Synthesis of biologically active influenza virus core proteins using a vaccinia virus-T7 RNA polymerase expression system. Journal of General Virology 75:2109–2114
    [Google Scholar]
  27. Mena I., Vivo A., Perez E., Portela A. 1996; Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids. Journal of Virology 70:5016–5024
    [Google Scholar]
  28. Mitnaul L. J., Castrucci M. R., Murti K. G., Kawaoka Y. 1996; The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication. Journal of Virology 70:873–879
    [Google Scholar]
  29. Murti K. G., Brown P. S., Bean W. J. Jr, Webster R. G. 1992; Composition of the helical internal components of influenza virus as revealed by immunogold labeling/electron microscopy. Virology 186:294–299
    [Google Scholar]
  30. Nemeroff M. E., Barabino S. M. L., 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. Molecular Cell 1:991–1000
    [Google Scholar]
  31. 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. Proceedings of the National Academy of Sciences, USA 87:5988–5992
    [Google Scholar]
  32. O’Neill R. E., Talon J., Palese P. 1998; The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO Journal 17:288–296
    [Google Scholar]
  33. Ortín J. 1998; Multiple levels of posttranscriptional regulation of influenza virus gene expression. Seminars in Virology 8:335–342
    [Google Scholar]
  34. Palese P., Tobita K., Ueda M., Compans R. W. 1974; Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61:397–410
    [Google Scholar]
  35. Pattnaik A. K., Wertz G. W. 1990; Replication and amplification of defective interfering particle RNAs of vesicular stomatitis virus in cells expressing viral proteins from vectors containing cloned cDNAs. Journal of Virology 64:2948–2957
    [Google Scholar]
  36. Pattnaik A. K., Wertz G. W. 1991; Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles. Proceedings of the National Academy of Sciences, USA 88:1379–1383
    [Google Scholar]
  37. Piccone M. E., Fernandez-Sesma A., Palese P. 1993; Mutational analysis of the influenza virus vRNA promoter. Virus Research 28:99–112
    [Google Scholar]
  38. Pinto L. H., Holsinger L. J., Lamb R. A. 1992; Influenza virus M2 protein has ion channel activity. Cell 69:517–528
    [Google Scholar]
  39. Sakaguchi T., Leser G. P., Lamb R. A. 1996; The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus. Journal of Cell Biology 133:733–747
    [Google Scholar]
  40. Sánchez-Fauquier A., Villanueva N., Melero J. A. 1987; Isolation of cross-reactive, subtype-specific monoclonal antibodies against influenza virus HA1 and HA2 hemagglutinin subunits. Archives of Virology 97:251–265
    [Google Scholar]
  41. Sanz-Ezquerro J. J., de la Luna S., Ortfn J., Nieto A. 1995; Individual expression of influenza virus PA protein induces degradation of coexpressed proteins. Journal of Virology 69:2420–2426
    [Google Scholar]
  42. Shih S. R., Nemeroff M. E., Krug R. M. 1995; The choice of alternative 5′ splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex. Proceedings of the National Academy of Sciences, USA 92:6324–6328
    [Google Scholar]
  43. Sugrue R. J., Bahadur G., Zambon M. C., Hall-Smith M., Douglas A. R., Hay A. J. 1990; Specific structural alteration of the influenza haemagglutinin by amantadine. EMBO Journal 9:3469–3476
    [Google Scholar]
  44. Teng M. N., Collins P. L. 1998; Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles. Journal of Virology 72:5707–5716
    [Google Scholar]
  45. Valcárcel J., Portela A., Ortfn J. 1991; Regulated M1 mRNA splicing in influenza virus-infected cells. Journal of General Virology 72:1301–1308
    [Google Scholar]
  46. Whittaker G., Bui M., Helenius A. 1996; The role of nuclear import and export in influenza virus infection. Trends in Cell Biology 6:67–71
    [Google Scholar]
  47. Yang P., Bansal A., Liu C., Air G. M. 1997; Hemagglutinin specificity and neuraminidase coding capacity of neuraminidase-deficient influenza viruses. Virology 229:155–165
    [Google Scholar]
  48. 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
    [Google Scholar]
  49. Zhirnov O. P. 1992; Isolation of matrix protein M1 from influenza viruses by acid-dependent extraction with nonionic detergent. Virology 186:324–330
    [Google Scholar]
  50. Zvonarjev A. Y., Ghendon Y. Z. 1980; Influence of membrane (M) protein on influenza A virus virion transcriptase activity in vitro and its susceptibility to rimantadine. Journal of Virology 33583–586
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-80-7-1635
Loading
/content/journal/jgv/10.1099/0022-1317-80-7-1635
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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