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. 1990 Oct;64(10):4978–4987. doi: 10.1128/jvi.64.10.4978-4987.1990

Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein.

P Dupraz 1, S Oertle 1, C Meric 1, P Damay 1, P F Spahr 1
PMCID: PMC247989  PMID: 2168981

Abstract

To extend our previous studies of the function of the Cys-His box of Rous sarcoma virus NC protein, we have constructed a series of point mutations of the conserved or nonconserved amino acids of the proximal Cys-His box and a one-amino-acid deletion. All mutants were characterized for production of viral proteins and particles, for packaging and maturation of viral RNA, for reverse transcriptase activity, and for infectivity. Our results indicated the following. (i) Mutations affecting the strictly conserved amino acids cysteine 21, cysteine 24, and histidine 29 were lethal; only the mutant His-29----Pro was still able to package viral RNA, most of it in an immature form. (ii) Mutation of the highly conserved glycine 28 to valine reduced viral RNA packaging by 90% and infectivity 30-fold, whereas mutant Gly-28----Ala was fully infectious. This suggests a steric hindrance limit at this position. (iii) Shortening the distance between cysteine 24 and histidine 29 by deleting one amino acid abolished the maturation of viral RNA and yielded noninfectious particles. (iv) Substitution of tyrosine 22 by serine lowered viral RNA packaging efficiency and yielded particles that were 400-fold less infectious; double mutant Tyr-22Thr-23----SerSer had the same infectivity as Tyr-22----Ser, whereas mutant Thr-23----Ser was fully infectious. (v) Changing glutamine 33 to a charged glutamate residue did not affect virus infectivity. Similarities and differences between our avian mutants and those in murine retroviruses are discussed.

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Selected References

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

  1. Aldovini A., Young R. A. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990 May;64(5):1920–1926. doi: 10.1128/jvi.64.5.1920-1926.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bender M. A., Palmer T. D., Gelinas R. E., Miller A. D. Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J Virol. 1987 May;61(5):1639–1646. doi: 10.1128/jvi.61.5.1639-1646.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  4. Bieth E., Gabus C., Darlix J. L. A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro. Nucleic Acids Res. 1990 Jan 11;18(1):119–127. doi: 10.1093/nar/18.1.119. [DOI] [PMC free article] [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. Darlix J. L., Spahr P. F. Binding sites of viral protein P19 onto Rous sarcoma virus RNA and possible controls of viral functions. J Mol Biol. 1982 Sep 15;160(2):147–161. doi: 10.1016/0022-2836(82)90172-3. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Fleissner E., Tress E. Isolation of a ribonucleoprotein structure from oncornaviruses. J Virol. 1973 Dec;12(6):1612–1615. doi: 10.1128/jvi.12.6.1612-1615.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Fu X., Phillips N., Jentoft J., Tuazon P. T., Traugh J. A., Leis J. Site-specific phosphorylation of avian retrovirus nucleocapsid protein pp12 regulates binding to viral RNA. Evidence for different protein conformations. J Biol Chem. 1985 Aug 15;260(17):9941–9947. [PubMed] [Google Scholar]
  11. Gallis B., Linial M., Eisenman R. An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA. Virology. 1979 Apr 15;94(1):146–161. doi: 10.1016/0042-6822(79)90445-8. [DOI] [PubMed] [Google Scholar]
  12. Gauss P., Krassa K. B., McPheeters D. S., Nelson M. A., Gold L. Zinc (II) and the single-stranded DNA binding protein of bacteriophage T4. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8515–8519. doi: 10.1073/pnas.84.23.8515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Giedroc D. P., Keating K. M., Williams K. R., Coleman J. E. The function of zinc in gene 32 protein from T4. Biochemistry. 1987 Aug 25;26(17):5251–5259. doi: 10.1021/bi00391a007. [DOI] [PubMed] [Google Scholar]
  14. Giedroc D. P., Keating K. M., Williams K. R., Konigsberg W. H., Coleman J. E. Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8452–8456. doi: 10.1073/pnas.83.22.8452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Goff S., Traktman P., Baltimore D. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J Virol. 1981 Apr;38(1):239–248. doi: 10.1128/jvi.38.1.239-248.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Green L. M., Berg J. M. A retroviral Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys peptide binds metal ions: spectroscopic studies and a proposed three-dimensional structure. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4047–4051. doi: 10.1073/pnas.86.11.4047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Katz R. A., Jentoft J. E. What is the role of the cys-his motif in retroviral nucleocapsid (NC) proteins? Bioessays. 1989 Dec;11(6):176–181. doi: 10.1002/bies.950110605. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kawai S., Koyama T. Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biological properties of mutant TK15 and mutant-induced transformants. J Virol. 1984 Jul;51(1):147–153. doi: 10.1128/jvi.51.1.147-153.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Koyama T., Harada F., Kawai S. Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biochemical properties of mutant TK15 and mutant-induced transformants. J Virol. 1984 Jul;51(1):154–162. doi: 10.1128/jvi.51.1.154-162.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. 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]
  32. Leis J., Johnson S., Collins L. S., Traugh J. A. Effects of phosphorylation of avian retrovirus nucleocapsid protein pp12 on binding of viral RNA. J Biol Chem. 1984 Jun 25;259(12):7726–7732. [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Mount S. M., Rubin G. M. Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins. Mol Cell Biol. 1985 Jul;5(7):1630–1638. doi: 10.1128/mcb.5.7.1630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Méric C., Darlix J. L., Spahr P. F. It is Rous sarcoma virus protein P12 and not P19 that binds tightly to Rous sarcoma virus RNA. J Mol Biol. 1984 Mar 15;173(4):531–538. doi: 10.1016/0022-2836(84)90396-6. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. 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]
  40. Prats A. C., Roy C., Wang P. A., Erard M., Housset V., Gabus C., Paoletti C., Darlix J. L. cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA. J Virol. 1990 Feb;64(2):774–783. doi: 10.1128/jvi.64.2.774-783.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Prats A. C., Sarih L., Gabus C., Litvak S., Keith G., Darlix J. L. Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO J. 1988 Jun;7(6):1777–1783. doi: 10.1002/j.1460-2075.1988.tb03008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Roberts W. J., Pan T., Elliott J. I., Coleman J. E., Williams K. R. p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39. Biochemistry. 1989 Dec 26;28(26):10043–10047. doi: 10.1021/bi00452a024. [DOI] [PubMed] [Google Scholar]
  43. Schiff L. A., Nibert M. L., Fields B. N. Characterization of a zinc blotting technique: evidence that a retroviral gag protein binds zinc. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4195–4199. doi: 10.1073/pnas.85.12.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Summers M. F., South T. L., Kim B., Hare D. R. High-resolution structure of an HIV zinc fingerlike domain via a new NMR-based distance geometry approach. Biochemistry. 1990 Jan 16;29(2):329–340. doi: 10.1021/bi00454a005. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. Williams K. R., LoPresti M. B., Setoguchi M., Konigsberg W. H. Amino acid sequence of the T4 DNA helix-destabilizing protein. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4614–4617. doi: 10.1073/pnas.77.8.4614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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