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Journal of Virology logoLink to Journal of Virology
. 1992 Aug;66(8):4662–4670. doi: 10.1128/jvi.66.8.4662-4670.1992

Specificity of Rous sarcoma virus nucleocapsid protein in genomic RNA packaging.

P Dupraz 1, P F Spahr 1
PMCID: PMC241290  PMID: 1378506

Abstract

Site-directed mutagenesis has shown that the nucleocapsid (NC) protein of Rous sarcoma virus (RSV) is required for packaging and dimerization of viral RNA. However, it has not been possible to demonstrate, in vivo or in vitro, specific binding of viral RNA sequences by NC. To determine whether specific packaging of viral RNA is mediated by NC in vivo, we have constructed RSV mutants carrying sequences of Moloney murine leukemia virus (MoMuLV). Either the NC coding region alone, the psi RNA packaging sequence, or both the NC and psi sequences of MoMuLV were substituted for the corresponding regions of a full-length RSV clone to yield chimeric plasmid pAPrcMNC, pAPrc psi M, or pAPrcM psi M, respectively. In addition, a mutant of RSV in which the NC is completely deleted was tested as a control. Upon transfection, each of the chimeric mutants produced viral particles containing processed core proteins but were noninfectious. Thus, MoMuLV NC can replace RSV NC functionally in the assembly and release of mature virions but not in infectivity. Surprisingly, the full-deletion mutant showed a strong block in virus release, suggesting that NC is involved in virus assembly. Mutant PrcMNC packaged 50- to 100-fold less RSV RNA than did the wild type; in cotransfection experiments, MoMuLV RNA was preferentially packaged. This result suggests that the specific recognition of viral RNA during virus assembly involves, at least in part, the NC protein.

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Selected References

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  1. Anderson D. J., Lee P., Levine K. L., Sang J. S., Shah S. A., Yang O. O., Shank P. R., Linial M. L. Molecular cloning and characterization of the RNA packaging-defective retrovirus SE21Q1b. J Virol. 1992 Jan;66(1):204–216. doi: 10.1128/jvi.66.1.204-216.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aronoff R., Linial M. Specificity of retroviral RNA packaging. J Virol. 1991 Jan;65(1):71–80. doi: 10.1128/jvi.65.1.71-80.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Canaani E., Helm K. V., Duesberg P. Evidence for 30-40S RNA as precursor of the 60-70S RNA of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1973 Feb;70(2):401–405. doi: 10.1073/pnas.70.2.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colicelli J., Goff S. P. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. doi: 10.1016/0022-2836(88)90378-6. [DOI] [PubMed] [Google Scholar]
  5. Covey S. N. Amino acid sequence homology in gag region of reverse transcribing elements and the coat protein gene of cauliflower mosaic virus. Nucleic Acids Res. 1986 Jan 24;14(2):623–633. doi: 10.1093/nar/14.2.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davis J., Scherer M., Tsai W. P., Long C. Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA. J Virol. 1976 May;18(2):709–718. doi: 10.1128/jvi.18.2.709-718.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dupraz P., Oertle S., Meric C., Damay P., Spahr P. F. Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein. J Virol. 1990 Oct;64(10):4978–4987. doi: 10.1128/jvi.64.10.4978-4987.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fu X. D., Katz R. A., Skalka A. M., Leis J. Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12. Mutation which affects RNA binding in vitro blocks viral replication. J Biol Chem. 1988 Feb 15;263(5):2140–2145. [PubMed] [Google Scholar]
  9. 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]
  10. Gorelick R. J., Henderson L. E., Hanser J. P., Rein A. Point mutants of Moloney murine leukemia virus that fail to package viral RNA: evidence for specific RNA recognition by a "zinc finger-like" protein sequence. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8420–8424. doi: 10.1073/pnas.85.22.8420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Göttlinger H. G., Dorfman T., Sodroski J. G., Haseltine W. A. Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3195–3199. doi: 10.1073/pnas.88.8.3195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jentoft J. E., Smith L. M., Fu X. D., Johnson M., Leis J. Conserved cysteine and histidine residues of the avian myeloblastosis virus nucleocapsid protein are essential for viral replication but are not "zinc-binding fingers". Proc Natl Acad Sci U S A. 1988 Oct;85(19):7094–7098. doi: 10.1073/pnas.85.19.7094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Karpel R. L., Henderson L. E., Oroszlan S. Interactions of retroviral structural proteins with single-stranded nucleic acids. J Biol Chem. 1987 Apr 15;262(11):4961–4967. [PubMed] [Google Scholar]
  14. Katoh I., Kyushiki H., Sakamoto Y., Ikawa Y., Yoshinaka Y. Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5'-terminal genomic RNA fragment. J Virol. 1991 Dec;65(12):6845–6855. doi: 10.1128/jvi.65.12.6845-6855.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Katz R. A., Terry R. W., Skalka A. M. A conserved cis-acting sequence in the 5' leader of avian sarcoma virus RNA is required for packaging. J Virol. 1986 Jul;59(1):163–167. doi: 10.1128/jvi.59.1.163-167.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Khandjian E. W., Méric C. A procedure for Northern blot analysis of native RNA. Anal Biochem. 1986 Nov 15;159(1):227–232. doi: 10.1016/0003-2697(86)90332-5. [DOI] [PubMed] [Google Scholar]
  17. Khandjian E. W. UV crosslinking of RNA to nylon membrane enhances hybridization signals. Mol Biol Rep. 1986;11(2):107–115. doi: 10.1007/BF00364822. [DOI] [PubMed] [Google Scholar]
  18. Kohl N. E., Diehl R. E., Rands E., Davis L. J., Hanobik M. G., Wolanski B., Dixon R. A. Expression of active human immunodeficiency virus type 1 protease by noninfectious chimeric virus particles. J Virol. 1991 Jun;65(6):3007–3014. doi: 10.1128/jvi.65.6.3007-3014.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leis J. P., McGinnis J., Green R. W. Rous sarcoma virus p19 binds to specific double-stranded regions of viral RNA: effect of p19 on cleavage of viral RNA by RNase III. Virology. 1978 Jan;84(1):87–98. doi: 10.1016/0042-6822(78)90220-9. [DOI] [PubMed] [Google Scholar]
  21. Leis J. P., Scheible P., Smith R. E. Correlation of RNA binding affinity of avian oncornavirus p19 proteins with the extent of processing of virus genome RNA in cells. J Virol. 1980 Sep;35(3):722–731. doi: 10.1128/jvi.35.3.722-731.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Leis J., Jentoft J. Characteristics and regulation of interaction of avian retrovirus pp12 protein with viral RNA. J Virol. 1983 Nov;48(2):361–369. doi: 10.1128/jvi.48.2.361-369.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Linial M. L., Miller A. D. Retroviral RNA packaging: sequence requirements and implications. Curr Top Microbiol Immunol. 1990;157:125–152. doi: 10.1007/978-3-642-75218-6_5. [DOI] [PubMed] [Google Scholar]
  24. Linial M., Medeiros E., Hayward W. S. An avian oncovirus mutant (SE 21Q1b) deficient in genomic RNA: biological and biochemical characterization. Cell. 1978 Dec;15(4):1371–1381. doi: 10.1016/0092-8674(78)90062-4. [DOI] [PubMed] [Google Scholar]
  25. Lo K. M., Jones S. S., Hackett N. R., Khorana H. G. Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2285–2289. doi: 10.1073/pnas.81.8.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lobel L. I., Goff S. P. Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: positions of viable insertion mutations. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4149–4153. doi: 10.1073/pnas.81.13.4149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Luban J., Goff S. P. Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein. J Virol. 1991 Jun;65(6):3203–3212. doi: 10.1128/jvi.65.6.3203-3212.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  29. Morikawa S., Booth T. F., Bishop D. H. Analyses of the requirements for the synthesis of virus-like particles by feline immunodeficiency virus gag using baculovirus vectors. Virology. 1991 Jul;183(1):288–297. doi: 10.1016/0042-6822(91)90141-w. [DOI] [PubMed] [Google Scholar]
  30. Murphy J. E., Goff S. P. Construction and analysis of deletion mutations in the U5 region of Moloney murine leukemia virus: effects on RNA packaging and reverse transcription. J Virol. 1989 Jan;63(1):319–327. doi: 10.1128/jvi.63.1.319-327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Méric C., Goff S. P. Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein. J Virol. 1989 Apr;63(4):1558–1568. doi: 10.1128/jvi.63.4.1558-1568.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Méric C., Gouilloud E., Spahr P. F. Mutations in Rous sarcoma virus nucleocapsid protein p12 (NC): deletions of Cys-His boxes. J Virol. 1988 Sep;62(9):3328–3333. doi: 10.1128/jvi.62.9.3328-3333.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Oertle S., Spahr P. F. Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA. J Virol. 1990 Dec;64(12):5757–5763. doi: 10.1128/jvi.64.12.5757-5763.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Royer M., Cerutti M., Gay B., Hong S. S., Devauchelle G., Boulanger P. Functional domains of HIV-1 gag-polyprotein expressed in baculovirus-infected cells. Virology. 1991 Sep;184(1):417–422. doi: 10.1016/0042-6822(91)90861-5. [DOI] [PubMed] [Google Scholar]
  36. Sen A., Sherr C. J., Todaro G. J. Phosphorylation of murine type C viral p12 proteins regulates their extent of binding to the homologous viral RNA. Cell. 1977 Mar;10(3):489–496. doi: 10.1016/0092-8674(77)90036-8. [DOI] [PubMed] [Google Scholar]
  37. Sen A., Sherr C. J., Todaro G. J. Specific binding of the type C viral core protein p12 with purified viral RNA. Cell. 1976 Jan;7(1):21–32. doi: 10.1016/0092-8674(76)90251-8. [DOI] [PubMed] [Google Scholar]
  38. Sen A., Todaro G. J. The genome-associated, specific RNA binding proteins of avian and mammalian type C viruses. Cell. 1977 Jan;10(1):91–99. doi: 10.1016/0092-8674(77)90143-x. [DOI] [PubMed] [Google Scholar]
  39. Shank P. R., Linial M. Avian oncovirus mutant (SE21Q1b) deficient in genomic RNA: characterization of a deletion in the provirus. J Virol. 1980 Nov;36(2):450–456. doi: 10.1128/jvi.36.2.450-456.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Smith B. J., Bailey J. M. The binding of an avian myeloblastosis virus basic 12,000 dalton protein to nucleic acids. Nucleic Acids Res. 1979 Dec 11;7(7):2055–2072. doi: 10.1093/nar/7.7.2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sorge J., Ricci W., Hughes S. H. cis-Acting RNA packaging locus in the 115-nucleotide direct repeat of Rous sarcoma virus. J Virol. 1983 Dec;48(3):667–675. doi: 10.1128/jvi.48.3.667-675.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Steeg C. M., Vogt V. M. RNA-binding properties of the matrix protein (p19gag) of avian sarcoma and leukemia viruses. J Virol. 1990 Feb;64(2):847–855. doi: 10.1128/jvi.64.2.847-855.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stewart L., Schatz G., Vogt V. M. Properties of avian retrovirus particles defective in viral protease. J Virol. 1990 Oct;64(10):5076–5092. doi: 10.1128/jvi.64.10.5076-5092.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sykora K. W., Moelling K. Properties of the avian viral protein p12. J Gen Virol. 1981 Aug;55(Pt 2):379–391. doi: 10.1099/0022-1317-55-2-379. [DOI] [PubMed] [Google Scholar]
  45. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  46. Weldon R. A., Jr, Erdie C. R., Oliver M. G., Wills J. W. Incorporation of chimeric gag protein into retroviral particles. J Virol. 1990 Sep;64(9):4169–4179. doi: 10.1128/jvi.64.9.4169-4179.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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