Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1997 Feb;71(2):918–924. doi: 10.1128/jvi.71.2.918-924.1997

An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis.

T M Myers 1, S A Moyer 1
PMCID: PMC191139  PMID: 8995608

Abstract

The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.

Full Text

The Full Text of this article is available as a PDF (434.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bass S. H., Mulkerrin M. G., Wells J. A. A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4498–4502. doi: 10.1073/pnas.88.10.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett W. F., Paoni N. F., Keyt B. A., Botstein D., Jones A. J., Presta L., Wurm F. M., Zoller M. J. High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem. 1991 Mar 15;266(8):5191–5201. [PubMed] [Google Scholar]
  3. Buchholz C. J., Retzler C., Homann H. E., Neubert W. J. The carboxy-terminal domain of Sendai virus nucleocapsid protein is involved in complex formation between phosphoprotein and nucleocapsid-like particles. Virology. 1994 Nov 1;204(2):770–776. doi: 10.1006/viro.1994.1592. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Calain P., Curran J., Kolakofsky D., Roux L. Molecular cloning of natural paramyxovirus copy-back defective interfering RNAs and their expression from DNA. Virology. 1992 Nov;191(1):62–71. doi: 10.1016/0042-6822(92)90166-m. [DOI] [PubMed] [Google Scholar]
  6. Calain P., Roux L. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol. 1993 Aug;67(8):4822–4830. doi: 10.1128/jvi.67.8.4822-4830.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carlsen S. R., Peluso R. W., Moyer S. A. In vitro replication of Sendai virus wild-type and defective interfering particle genome RNAs. J Virol. 1985 May;54(2):493–500. doi: 10.1128/jvi.54.2.493-500.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chandrika R., Horikami S. M., Smallwood S., Moyer S. A. Mutations in conserved domain I of the Sendai virus L polymerase protein uncouple transcription and replication. Virology. 1995 Nov 10;213(2):352–363. doi: 10.1006/viro.1995.0008. [DOI] [PubMed] [Google Scholar]
  9. Chandrika R., Myers T., Moyer S. A. Measles virus nucleocapsid protein can function in Sendai virus defective interfering particle genome synthesis in vitro. Virology. 1995 Jan 10;206(1):777–782. doi: 10.1016/s0042-6822(95)80009-3. [DOI] [PubMed] [Google Scholar]
  10. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Curran J., Homann H., Buchholz C., Rochat S., Neubert W., Kolakofsky D. The hypervariable C-terminal tail of the Sendai paramyxovirus nucleocapsid protein is required for template function but not for RNA encapsidation. J Virol. 1993 Jul;67(7):4358–4364. doi: 10.1128/jvi.67.7.4358-4364.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Diamond S. E., Kirkegaard K. Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis. J Virol. 1994 Feb;68(2):863–876. doi: 10.1128/jvi.68.2.863-876.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hassett D. E., Condit R. C. Targeted construction of temperature-sensitive mutations in vaccinia virus by replacing clustered charged residues with alanine. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4554–4558. doi: 10.1073/pnas.91.10.4554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heggeness M. H., Scheid A., Choppin P. W. Conformation of the helical nucleocapsids of paramyxoviruses and vesicular stomatitis virus: reversible coiling and uncoiling induced by changes in salt concentration. Proc Natl Acad Sci U S A. 1980 May;77(5):2631–2635. doi: 10.1073/pnas.77.5.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Homann H. E., Willenbrink W., Buchholz C. J., Neubert W. J. Sendai virus protein-protein interactions studied by a protein-blotting protein-overlay technique: mapping of domains on NP protein required for binding to P protein. J Virol. 1991 Mar;65(3):1304–1309. doi: 10.1128/jvi.65.3.1304-1309.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Horikami S. M., Moyer S. A. Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro. Virology. 1995 Aug 20;211(2):577–582. doi: 10.1006/viro.1995.1440. [DOI] [PubMed] [Google Scholar]
  22. Hudson L. D., Condra C., Lazzarini R. A. Cloning and expression of a viral phosphoprotein: structure suggests vesicular stomatitis virus NS may function by mimicking an RNA template. J Gen Virol. 1986 Aug;67(Pt 8):1571–1579. doi: 10.1099/0022-1317-67-8-1571. [DOI] [PubMed] [Google Scholar]
  23. Kolakofsky D. Isolation and characterization of Sendai virus DI-RNAs. Cell. 1976 Aug;8(4):547–555. doi: 10.1016/0092-8674(76)90223-3. [DOI] [PubMed] [Google Scholar]
  24. Miyahara K., Kitada S., Yoshimoto M., Matsumura H., Kawano M., Komada H., Tsurudome M., Kusagawa S., Nishio M., Ito Y. Molecular evolution of human paramyxoviruses. Nucleotide sequence analyses of the human parainfluenza type 1 virus NP and M protein genes and construction of phylogenetic trees for all the human paramyxoviruses. Arch Virol. 1992;124(3-4):255–268. doi: 10.1007/BF01309807. [DOI] [PubMed] [Google Scholar]
  25. Morgan E. M., Re G. G., Kingsbury D. W. Complete sequence of the Sendai virus NP gene from a cloned insert. Virology. 1984 May;135(1):279–287. doi: 10.1016/0042-6822(84)90137-5. [DOI] [PubMed] [Google Scholar]
  26. Pattnaik A. K., Ball L. A., LeGrone A. W., Wertz G. W. Infectious defective interfering particles of VSV from transcripts of a cDNA clone. Cell. 1992 Jun 12;69(6):1011–1020. doi: 10.1016/0092-8674(92)90619-n. [DOI] [PubMed] [Google Scholar]
  27. Portner A., Murti K. G. Localization of P, NP, and M proteins on Sendai virus nucleocapsid using immunogold labeling. Virology. 1986 Apr 30;150(2):469–478. doi: 10.1016/0042-6822(86)90311-9. [DOI] [PubMed] [Google Scholar]
  28. Portner A., Murti K. G., Morgan E. M., Kingsbury D. W. Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy. Virology. 1988 Mar;163(1):236–239. doi: 10.1016/0042-6822(88)90257-7. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Shioda T., Hidaka Y., Kanda T., Shibuta H., Nomoto A., Iwasaki K. Sequence of 3,687 nucleotides from the 3' end of Sendai virus genome RNA and the predicted amino acid sequences of viral NP, P and C proteins. Nucleic Acids Res. 1983 Nov 11;11(21):7317–7330. doi: 10.1093/nar/11.21.7317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wells J. A., Cunningham B. C., Fuh G., Lowman H. B., Bass S. H., Mulkerrin M. G., Ultsch M., deVos A. M. The molecular basis for growth hormone-receptor interactions. Recent Prog Horm Res. 1993;48:253–275. [PubMed] [Google Scholar]
  32. Wertman K. F., Drubin D. G., Botstein D. Systematic mutational analysis of the yeast ACT1 gene. Genetics. 1992 Oct;132(2):337–350. doi: 10.1093/genetics/132.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES