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. 1979 Oct;32(1):304–313. doi: 10.1128/jvi.32.1.304-313.1979

Assembly of vesicular stomatitis virus nucleocapsids in vivo: a kinetic analysis.

C H Hsu, D W Kingsbury, K G Murti
PMCID: PMC353554  PMID: 232181

Abstract

Pulse-chase labeling and cell fractionation were used to examine the pathways taken by the three nucleocapsid polypeptide species of vesicular stomatitis virus into nucleocapsids and then into virions. An improved method of polyacrylamide gel electrophoresis resolved nucleocapsid polypeptides N and NS from cellular actin, facilitating accurate quantitation of the viral polypeptides. Contrary to previous belief, the rate of NS synthesis was found to be a constant fraction of total virus protein synthesis throughout infection, indicating a consistent mechanism of virus protein synthesis regulation. In the kinetic studies, each polypeptide species displayed the following characteristic behavior. (i) Structural polypeptide N was the only species that entered a metabolically active soluble pool before assembly into nucleocapsids. The size of this pool increased with time after infection, causing an increasing delay in the appearance of pulse-labeled N molecules in nucleocapsids. (ii) Throughout infection, the entire complement of L molecules entered nucleocapsids immediately after their synthesis, without diversion through a soluble pool. (iii) Although 75% of newly synthesized molecules of the transcriptase-associated protein NS entered a soluble pool, they never emerged from the compartment. At all times after infection, about 25% of the NS molecules bypassed the soluble pool and entered nucleocapsids directly after their synthesis, as if in concert with L. These results indicate that VSV nucleocapsid assembly in vivo is a stepwise process, comprising an initial condensation of N with the viral RNA, followed by attachment of L and NS, analogous to the stepwise assembly of Sendai virus nucleocapsids. (D. W. Kingsbury, C.-H. Hsu, and K. G. Murti. Virology 91:86-94, 1978). About half of the intracellular nucleocapsids were recovered in a form that sedimented at anomalously low centrifugal forces, reflecting an association with large cellular organelles. This attachment was mediated mainly by electrostatic forces, since these "bound" nucleocapsids were released by elevated salt concentrations. The kinetic behavior of nucleocapsid polypeptides was the same in both fractions, providing no evidence for a division of nucleocapsid functions between cellular compartments.

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Selected References

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

  1. Atkinson P. H., Moyer S. A., Summers D. F. Assembly of vesicular stomatitis virus glycoprotein and matrix protein into HeLa cell plasma membranes. J Mol Biol. 1976 Apr 15;102(3):613–631. doi: 10.1016/0022-2836(76)90338-7. [DOI] [PubMed] [Google Scholar]
  2. Ball L. A. Transcriptional mapping of vesicular stomatitis virus in vivo. J Virol. 1977 Jan;21(1):411–414. doi: 10.1128/jvi.21.1.411-414.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. CASPAR D. L., KLUG A. Physical principles in the construction of regular viruses. Cold Spring Harb Symp Quant Biol. 1962;27:1–24. doi: 10.1101/sqb.1962.027.001.005. [DOI] [PubMed] [Google Scholar]
  4. Cartwright B. The distribution of virus proteins in BHK 21 cells infected with vesicular stomatitis virus (Indiana C). J Gen Virol. 1973 Nov;21(2):407–411. doi: 10.1099/0022-1317-21-2-407. [DOI] [PubMed] [Google Scholar]
  5. Clinton G. M., Burge B. W., Huang A. S. Effects of phosphorylation and pH on the association of NS protein with vesicular stomatitis virus cores. J Virol. 1978 Aug;27(2):340–346. doi: 10.1128/jvi.27.2.340-346.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen G. H., Atkinson P. H., Summers D. F. Interactions of vesicular stomatitis virus structural proteins with HeLa plasma membranes. Nat New Biol. 1971 May 26;231(21):121–123. doi: 10.1038/newbio231121a0. [DOI] [PubMed] [Google Scholar]
  7. Coward J. E., Harter D. H., Hsu K. C., Morgan C. Electron microscopic study of development of vesicular stomatitis virus using ferritin-labelled antibodies. J Gen Virol. 1971 Oct;13(1):27–34. doi: 10.1099/0022-1317-13-1-27. [DOI] [PubMed] [Google Scholar]
  8. David A. E. Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane. J Mol Biol. 1973 May 5;76(1):135–148. doi: 10.1016/0022-2836(73)90085-5. [DOI] [PubMed] [Google Scholar]
  9. Emerson S. U., Wagner R. R. Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. J Virol. 1972 Aug;10(2):297–309. doi: 10.1128/jvi.10.2.297-309.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Emerson S. U., Yu Y. Both NS and L proteins are required for in vitro RNA synthesis by vesicular stomatitis virus. J Virol. 1975 Jun;15(6):1348–1356. doi: 10.1128/jvi.15.6.1348-1356.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kang C. Y., Prevec L. Proteins of vesicular stomatitis virus. 3. Intracellular synthesis and extracellular appearance of virus-specific proteins. Virology. 1971 Dec;46(3):678–690. doi: 10.1016/0042-6822(71)90070-5. [DOI] [PubMed] [Google Scholar]
  12. Kiley M. P., Wagner R. R. Ribonucleic acid species of intracellular nucleocapsids and released virions of vesicular stomatitis virus. J Virol. 1972 Aug;10(2):244–255. doi: 10.1128/jvi.10.2.244-255.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kingsbury D. W., Hsu C. H., Murti K. G. Intracellular metabolism of sendai virus nucleocapside. Virology. 1978 Nov;91(1):86–94. doi: 10.1016/0042-6822(78)90357-4. [DOI] [PubMed] [Google Scholar]
  14. Knipe D. M., Baltimore D., Lodish H. F. Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus. J Virol. 1977 Mar;21(3):1128–1139. doi: 10.1128/jvi.21.3.1128-1139.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Mellon M. G., Emerson S. U. Rebinding of transcriptase components (L and NS proteins) to the nucleocapsid template of vesicular stomatitis virus. J Virol. 1978 Sep;27(3):560–567. doi: 10.1128/jvi.27.3.560-567.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mudd J. A., Summers D. F. Protein synthesis in vesicular stomatitis virus-infected HeLa cells. Virology. 1970 Oct;42(2):328–340. doi: 10.1016/0042-6822(70)90277-1. [DOI] [PubMed] [Google Scholar]
  18. Naito S., Ishihama A. Function and structure of RNA polymerase from vesicular stomatitis virus. J Biol Chem. 1976 Jul 25;251(14):4307–4314. [PubMed] [Google Scholar]
  19. Petric M., Prevec L. Vesicular stomatitis virus--a new interfering particle, intracellular structures, and virus-specific RNA. Virology. 1970 Aug;41(4):615–630. doi: 10.1016/0042-6822(70)90427-7. [DOI] [PubMed] [Google Scholar]
  20. Portner A. Association of nucleocapsid polypeptides with defective RNA synthesis in a temperature-sensitive mutant of Sendai virus. Virology. 1977 Apr;77(2):481–489. doi: 10.1016/0042-6822(77)90473-1. [DOI] [PubMed] [Google Scholar]
  21. Portner A., Kingsbury D. W. Regulatory events in the synthesis of Sendai virus polypeptides and their assembly into virions. Virology. 1976 Aug;73(1):79–88. doi: 10.1016/0042-6822(76)90062-3. [DOI] [PubMed] [Google Scholar]
  22. Rothman J. E., Lodish H. F. Synchronised transmembrane insertion and glycosylation of a nascent membrane protein. Nature. 1977 Oct 27;269(5631):775–780. doi: 10.1038/269775a0. [DOI] [PubMed] [Google Scholar]
  23. Soria M., Little S. P., Huang A. S. Characterization of vesicular stomatitis virus nucleocapsids. I. Complementary 40 S RNA molecules in nucleocapsids. Virology. 1974 Sep;61(1):270–280. doi: 10.1016/0042-6822(74)90261-x. [DOI] [PubMed] [Google Scholar]
  24. Wagner R. R., Kiley M. P., Snyder R. M., Schnaitman C. A. Cytoplasmic compartmentalization of the protein and ribonucleic acid species of vesicular stomatitis virus. J Virol. 1972 Apr;9(4):672–683. doi: 10.1128/jvi.9.4.672-683.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wagner R. R., Snyder R. M., Yamazaki S. Proteins of vesicular stomatitis virus: kinetics and cellular sites of synthesis. J Virol. 1970 May;5(5):548–558. doi: 10.1128/jvi.5.5.548-558.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zajac B. A., Hummeler K. Identification of vesicular stomatitis virus nucleoprotein in situ. J Gen Virol. 1971 Nov;13(2):215–220. doi: 10.1099/0022-1317-13-2-215. [DOI] [PubMed] [Google Scholar]

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