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. 1990 Nov;64(11):5628–5632. doi: 10.1128/jvi.64.11.5628-5632.1990

Rous sarcoma virus expression in Saccharomyces cerevisiae: processing and membrane targeting of the gag gene product.

D Bonnet 1, P F Spahr 1
PMCID: PMC248618  PMID: 2170688

Abstract

In avian cells, the product of the gag gene of Rous sarcoma virus, Pr76gag, has been shown to be targeted to the plasma membrane, to form virus particles, and then to be processed into mature viral gag proteins. To explore how these phenomena may be dependent upon cellular (host) factors, we expressed the Rous sarcoma virus gag gene in a lower eucaryote, Saccharomyces cerevisiae, and studied the behavior of the gag gene product. We show here that Pr76gag is processed in yeast cells and that this processing is specific, since it is abolished in a mutant in which the active site of the gag protease has been destroyed. In this mutant, the uncleaved precursor is found associated with the yeast plasma membrane, yet no virus particles were detected in cells or in the culture medium. From our results, we can speculate either that in yeast cells, a host protease initiates Pr76gag processing in the cytosol or that in avian cells, an inhibitor prevents the processing until the viral particle is formed.

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Selected References

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  1. Bolognesi D. P., Montelaro R. C., Frank H., Schäfer W. Assembly of type C oncornaviruses: a model. Science. 1978 Jan 13;199(4325):183–186. doi: 10.1126/science.202022. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Damsky C. H., Sheffield J. B., Tuszynski G. P., Warren L. Is there a role for actin in virus budding? J Cell Biol. 1977 Nov;75(2 Pt 1):593–605. doi: 10.1083/jcb.75.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Davis N. L., Rueckert R. R. Properties of a ribonucleoprotein particle isolated from Nonidet P-40-treated Rous sarcoma virus. J Virol. 1972 Nov;10(5):1010–1020. doi: 10.1128/jvi.10.5.1010-1020.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Edbauer C. A., Naso R. B. Cytoskeleton-associated Pr65gag and assembly of retrovirus temperature-sensitive mutants in chronically infected cells. Virology. 1984 Apr 30;134(2):389–397. doi: 10.1016/0042-6822(84)90306-4. [DOI] [PubMed] [Google Scholar]
  6. Felsenstein K. M., Goff S. P. Expression of the gag-pol fusion protein of Moloney murine leukemia virus without gag protein does not induce virion formation or proteolytic processing. J Virol. 1988 Jun;62(6):2179–2182. doi: 10.1128/jvi.62.6.2179-2182.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gheysen D., Jacobs E., de Foresta F., Thiriart C., Francotte M., Thines D., De Wilde M. Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells. Cell. 1989 Oct 6;59(1):103–112. doi: 10.1016/0092-8674(89)90873-8. [DOI] [PubMed] [Google Scholar]
  8. Goff S. P., Lobel L. I. Mutants of murine leukemia viruses and retroviral replication. Biochim Biophys Acta. 1987 Jul 8;907(2):93–123. doi: 10.1016/0304-419x(87)90001-1. [DOI] [PubMed] [Google Scholar]
  9. Goldfine H., Harley J. B., Wyke J. A. Effects of inhibitors of lipid synthesis on the replication of Rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins. Biochim Biophys Acta. 1978 Sep 22;512(2):229–240. doi: 10.1016/0005-2736(78)90249-3. [DOI] [PubMed] [Google Scholar]
  10. Hayman M. J., Hunter E., Vogt P. K. Inhibition of avian sarcoma virus replication by glucosamine: a specific effect on the synthesis and processing of viral proteins. Virology. 1976 Jun;71(2):402–411. doi: 10.1016/0042-6822(76)90368-8. [DOI] [PubMed] [Google Scholar]
  11. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobs E., Gheysen D., Thines D., Francotte M., de Wilde M. The HIV-1 Gag precursor Pr55gag synthesized in yeast is myristoylated and targeted to the plasma membrane. Gene. 1989 Jun 30;79(1):71–81. doi: 10.1016/0378-1119(89)90093-0. [DOI] [PubMed] [Google Scholar]
  13. Kotler M., Katz R. A., Skalka A. M. Activity of avian retroviral protease expressed in Escherichia coli. J Virol. 1988 Aug;62(8):2696–2700. doi: 10.1128/jvi.62.8.2696-2700.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kramer R. A., Schaber M. D., Skalka A. M., Ganguly K., Wong-Staal F., Reddy E. P. HTLV-III gag protein is processed in yeast cells by the virus pol-protease. Science. 1986 Mar 28;231(4745):1580–1584. doi: 10.1126/science.2420008. [DOI] [PubMed] [Google Scholar]
  15. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Le Grice S. F., Mills J., Mous J. Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation. EMBO J. 1988 Aug;7(8):2547–2553. doi: 10.1002/j.1460-2075.1988.tb03103.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Leal-Morales C. A., Bracker C. E., Bartnicki-Garcia S. Localization of chitin synthetase in cell-free homogenates of Saccharomyces cerevisiae: chitosomes and plasma membrane. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8516–8520. doi: 10.1073/pnas.85.22.8516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leis J., Baltimore D., Bishop J. M., Coffin J., Fleissner E., Goff S. P., Oroszlan S., Robinson H., Skalka A. M., Temin H. M. Standardized and simplified nomenclature for proteins common to all retroviruses. J Virol. 1988 May;62(5):1808–1809. doi: 10.1128/jvi.62.5.1808-1809.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mermer B., Malamy M., Coffin J. M. Rous sarcoma virus contains sequences which permit expression of the gag gene in Escherichia coli. Mol Cell Biol. 1983 Oct;3(10):1746–1758. doi: 10.1128/mcb.3.10.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Méric C., Spahr P. F. Rous sarcoma virus nucleic acid-binding protein p12 is necessary for viral 70S RNA dimer formation and packaging. J Virol. 1986 Nov;60(2):450–459. doi: 10.1128/jvi.60.2.450-459.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pepinsky R. B., Vogt V. M. Fine-structure analyses of lipid-protein and protein-protein interactions of gag protein p19 of the avian sarcoma and leukemia viruses by cyanogen bromide mapping. J Virol. 1984 Oct;52(1):145–153. doi: 10.1128/jvi.52.1.145-153.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pepinsky R. B., Vogt V. M. Identification of retrovirus matrix proteins by lipid-protein cross-linking. J Mol Biol. 1979 Jul 15;131(4):819–837. doi: 10.1016/0022-2836(79)90203-1. [DOI] [PubMed] [Google Scholar]
  24. Rein A., McClure M. R., Rice N. R., Luftig R. B., Schultz A. M. Myristylation site in Pr65gag is essential for virus particle formation by Moloney murine leukemia virus. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7246–7250. doi: 10.1073/pnas.83.19.7246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schaber M. D., DeChiara T. M., Kramer R. A. Yeast vectors for production of interferon. Methods Enzymol. 1986;119:416–423. doi: 10.1016/0076-6879(86)19061-6. [DOI] [PubMed] [Google Scholar]
  26. Schwartz D. E., Tizard R., Gilbert W. Nucleotide sequence of Rous sarcoma virus. Cell. 1983 Mar;32(3):853–869. doi: 10.1016/0092-8674(83)90071-5. [DOI] [PubMed] [Google Scholar]
  27. Scott J. H., Schekman R. Lyticase: endoglucanase and protease activities that act together in yeast cell lysis. J Bacteriol. 1980 May;142(2):414–423. doi: 10.1128/jb.142.2.414-423.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Skalka A. M. Retroviral proteases: first glimpses at the anatomy of a processing machine. Cell. 1989 Mar 24;56(6):911–913. doi: 10.1016/0092-8674(89)90621-1. [DOI] [PubMed] [Google Scholar]
  29. Vogt V. M., Bruckenstein D. A., Bell A. P. Avian sarcoma virus gag precursor polypeptide is not processed in mammalian cells. J Virol. 1982 Nov;44(2):725–730. doi: 10.1128/jvi.44.2.725-730.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vogt V. M., Eisenman R., Diggelmann H. Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide. J Mol Biol. 1975 Aug 15;96(3):471–493. doi: 10.1016/0022-2836(75)90174-6. [DOI] [PubMed] [Google Scholar]
  31. Welten-Verstegen G. W., Boer P., Steyn-Parvé E. P. Lipid-mediated glycosylation of endogenous proteins in isolated plasma membrane of Saccharomyces cerevisiae. J Bacteriol. 1980 Jan;141(1):342–349. doi: 10.1128/jb.141.1.342-349.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wills J. W., Craven R. C., Achacoso J. A. Creation and expression of myristylated forms of Rous sarcoma virus gag protein in mammalian cells. J Virol. 1989 Oct;63(10):4331–4343. doi: 10.1128/jvi.63.10.4331-4343.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Witte O. N., Baltimore D. Relationship of retrovirus polyprotein cleavages to virion maturation studied with temperature-sensitive murine leukemia virus mutants. J Virol. 1978 Jun;26(3):750–761. doi: 10.1128/jvi.26.3.750-761.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

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