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. 1995 Dec;69(12):8001–8010. doi: 10.1128/jvi.69.12.8001-8010.1995

Inducible expression of the P, V, and NP genes of the paramyxovirus simian virus 5 in cell lines and an examination of NP-P and NP-V interactions.

B Precious 1, D F Young 1, A Bermingham 1, R Fearns 1, M Ryan 1, R E Randall 1
PMCID: PMC189745  PMID: 7494313

Abstract

The P, V, and NP genes of the paramyxovirus simian virus 5 (SV5) were cloned such that their expression was regulated by the tetracycline-controlled transactivator (M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992), and mammalian cell lines that inducibly expressed individually the P, V, or NP protein or coexpressed the P plus NP or V plus NP proteins were isolated. A plasmid that expresses the tetracycline-controlled transactivator linked, via the foot-and-mouth disease virus 2A cleavage peptide sequence, to the neomycin aminoglycoside phosphotransferase gene was constructed. Cells were cotransfected with this plasmid, and the appropriate responder plasmids and clonies were selected on the basis of their resistance to Geneticin (via the neomycin aminoglycoside phosphotransferase gene). The properties of these cell lines, in terms of the induction of the P, V, and NP genes, are described in detail. Both the P and V proteins were phosphorylated when expressed alone. In immunoprecipitation studies using a monoclonal antibody that recognizes both the P and V proteins, a nonphosphorylated host cell protein with an estimated molecular weight of 150,000 was coprecipitated with V but not P. Immunofluorescence data demonstrated that when expressed separately, the P protein had a diffuse cytoplasmic distribution, but the related V protein had both a nuclear and cytoplasmic distribution. The NP protein had a granular cytoplasmic distribution, giving rise to punctate and granular fluorescence. Coexpression of the NP and P proteins resulted in the accumulation of large cytoplasmic inclusion aggregates, similar to those visualized at late times in SV5-infected cells. Coexpression of V with NP led to a partial redistribution of the NP protein in that the NP protein had both a diffuse cytoplasmic and nuclear distribution in the presence of V, but no NP-V aggregates or inclusion bodies were visualized. Direct binding studies also revealed that NP bound to both P and V. For SV5, these studies suggest that V may have a role in keeping NP soluble prior to encapsidation.

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Selected References

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  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buchholz C. J., Spehner D., Drillien R., Neubert W. J., Homann H. E. The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly. J Virol. 1993 Oct;67(10):5803–5812. doi: 10.1128/jvi.67.10.5803-5812.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chenik M., Chebli K., Gaudin Y., Blondel D. In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N): existence of two N-binding sites on P protein. J Gen Virol. 1994 Nov;75(Pt 11):2889–2896. doi: 10.1099/0022-1317-75-11-2889. [DOI] [PubMed] [Google Scholar]
  4. Curran J., Boeck R., Kolakofsky D. The Sendai virus P gene expresses both an essential protein and an inhibitor of RNA synthesis by shuffling modules via mRNA editing. EMBO J. 1991 Oct;10(10):3079–3085. doi: 10.1002/j.1460-2075.1991.tb07860.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Curran J., Marq J. B., Kolakofsky D. An N-terminal domain of the Sendai paramyxovirus P protein acts as a chaperone for the NP protein during the nascent chain assembly step of genome replication. J Virol. 1995 Feb;69(2):849–855. doi: 10.1128/jvi.69.2.849-855.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Curran J., Pelet T., Kolakofsky D. An acidic activation-like domain of the Sendai virus P protein is required for RNA synthesis and encapsidation. Virology. 1994 Aug 1;202(2):875–884. doi: 10.1006/viro.1994.1409. [DOI] [PubMed] [Google Scholar]
  7. Curran J., de Melo M., Moyer S., Kolakofsky D. Characterization of the Sendai virus V protein with an anti-peptide antiserum. Virology. 1991 Sep;184(1):108–116. doi: 10.1016/0042-6822(91)90827-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Einberger H., Mertz R., Hofschneider P. H., Neubert W. J. Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro. J Virol. 1990 Sep;64(9):4274–4280. doi: 10.1128/jvi.64.9.4274-4280.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fearns R., Young D. F., Randall R. E. Evidence that the paramyxovirus simian virus 5 can establish quiescent infections by remaining inactive in cytoplasmic inclusion bodies. J Gen Virol. 1994 Dec;75(Pt 12):3525–3539. doi: 10.1099/0022-1317-75-12-3525. [DOI] [PubMed] [Google Scholar]
  10. Fooks A. R., Stephenson J. R., Warnes A., Dowsett A. B., Rima B. K., Wilkinson G. W. Measles virus nucleocapsid protein expressed in insect cells assembles into nucleocapsid-like structures. J Gen Virol. 1993 Jul;74(Pt 7):1439–1444. doi: 10.1099/0022-1317-74-7-1439. [DOI] [PubMed] [Google Scholar]
  11. García J., García-Barreno B., Vivo A., Melero J. A. Cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22K protein. Virology. 1993 Jul;195(1):243–247. doi: 10.1006/viro.1993.1366. [DOI] [PubMed] [Google Scholar]
  12. Gossen M., Bonin A. L., Bujard H. Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci. 1993 Dec;18(12):471–475. doi: 10.1016/0968-0004(93)90009-c. [DOI] [PubMed] [Google Scholar]
  13. Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamaguchi M., Yoshida T., Nishikawa K., Naruse H., Nagai Y. Transcriptive complex of Newcastle disease virus. I. Both L and P proteins are required to constitute an active complex. Virology. 1983 Jul 15;128(1):105–117. doi: 10.1016/0042-6822(83)90322-7. [DOI] [PubMed] [Google Scholar]
  15. Hillen W., Berens C. Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu Rev Microbiol. 1994;48:345–369. doi: 10.1146/annurev.mi.48.100194.002021. [DOI] [PubMed] [Google Scholar]
  16. Horikami S. M., Curran J., Kolakofsky D., Moyer S. A. Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro. J Virol. 1992 Aug;66(8):4901–4908. doi: 10.1128/jvi.66.8.4901-4908.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kafatos F. C., Efstratiadis A., Forget B. G., Weissman S. M. Molecular evolution of human and rabbit beta-globin mRNAs. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5618–5622. doi: 10.1073/pnas.74.12.5618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  19. Liston P., Briedis D. J. Measles virus V protein binds zinc. Virology. 1994 Jan;198(1):399–404. doi: 10.1006/viro.1994.1050. [DOI] [PubMed] [Google Scholar]
  20. Markwell M. A., Fox C. F. Protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking. J Virol. 1980 Jan;33(1):152–166. doi: 10.1128/jvi.33.1.152-166.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Parks G. D., Ward C. D., Lamb R. A. Molecular cloning of the NP and L genes of simian virus 5: identification of highly conserved domains in paramyxovirus NP and L proteins. Virus Res. 1992 Mar;22(3):259–279. doi: 10.1016/0168-1702(92)90057-g. [DOI] [PubMed] [Google Scholar]
  22. Paterson R. G., Leser G. P., Shaughnessy M. A., Lamb R. A. The paramyxovirus SV5 V protein binds two atoms of zinc and is a structural component of virions. Virology. 1995 Apr 1;208(1):121–131. doi: 10.1006/viro.1995.1135. [DOI] [PubMed] [Google Scholar]
  23. Randall R. E., Dinwoodie N. Intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that ICP 4 is associated with progeny virus DNA. J Gen Virol. 1986 Oct;67(Pt 10):2163–2177. doi: 10.1099/0022-1317-67-10-2163. [DOI] [PubMed] [Google Scholar]
  24. Randall R. E., Young D. F., Goswami K. K., Russell W. C. Isolation and characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between human, canine and simian isolates. J Gen Virol. 1987 Nov;68(Pt 11):2769–2780. doi: 10.1099/0022-1317-68-11-2769. [DOI] [PubMed] [Google Scholar]
  25. Ryan K. W., Portner A., Murti K. G. Antibodies to paramyxovirus nucleoproteins define regions important for immunogenicity and nucleocapsid assembly. Virology. 1993 Mar;193(1):376–384. doi: 10.1006/viro.1993.1134. [DOI] [PubMed] [Google Scholar]
  26. Ryan M. D., Drew J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 1994 Feb 15;13(4):928–933. doi: 10.1002/j.1460-2075.1994.tb06337.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Southern J. A., Young D. F., Heaney F., Baumgärtner W. K., Randall R. E. Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics. J Gen Virol. 1991 Jul;72(Pt 7):1551–1557. doi: 10.1099/0022-1317-72-7-1551. [DOI] [PubMed] [Google Scholar]
  28. Spehner D., Kirn A., Drillien R. Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein. J Virol. 1991 Nov;65(11):6296–6300. doi: 10.1128/jvi.65.11.6296-6300.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas S. M., Lamb R. A., Paterson R. G. Two mRNAs that differ by two nontemplated nucleotides encode the amino coterminal proteins P and V of the paramyxovirus SV5. Cell. 1988 Sep 9;54(6):891–902. doi: 10.1016/S0092-8674(88)91285-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

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