• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. Apr 1989; 63(4): 1558–1568.
PMCID: PMC248388

Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys-His box of the nucleocapsid protein.

Abstract

To study the function of the retroviral nucleocapsid protein (NC), we have constructed point mutations in the gag gene of Moloney murine leukemia virus (MuLV) that affect a conserved cysteine-histidine motif of NC. The mutants were characterized biologically and biochemically. Cell lines producing the mutant virions were constructed in NIH 3T3 and rat2 cells, and the viral particles released by these cells were characterized for protein and RNA content. The results indicated that most mutations block replication and specifically inhibit the packaging of the MuLV genomic RNA. In some of the mutants, the packaging of the endogenous rat VL30 RNA was not affected as profoundly as was MuLV RNA. NC also seems to have another function distinct from dimer formation and packaging: one mutation reduced viral RNA packaging by only fivefold but completely abolished viral cDNA synthesis, suggesting a defect in reverse transcription.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Berg JM. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. [PubMed]
  • Bolognesi DP, Montelaro RC, Frank H, Schäfer W. Assembly of type C oncornaviruses: a model. Science. 1978 Jan 13;199(4325):183–186. [PubMed]
  • Bond VC, Wold B. Poly-L-ornithine-mediated transformation of mammalian cells. Mol Cell Biol. 1987 Jun;7(6):2286–2293. [PMC free article] [PubMed]
  • Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. [PubMed]
  • Colicelli J, Goff SP. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. [PubMed]
  • Copeland TD, Morgan MA, Oroszlan S. Complete amino acid sequence of the basic nucleic acid binding protein of feline leukemia virus. Virology. 1984 Feb;133(1):137–145. [PubMed]
  • Covey SN. 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. [PMC free article] [PubMed]
  • Crawford S, Goff SP. Mutations in gag proteins P12 and P15 of Moloney murine leukemia virus block early stages of infection. J Virol. 1984 Mar;49(3):909–917. [PMC free article] [PubMed]
  • Davis J, Scherer M, Tsai WP, 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. [PMC free article] [PubMed]
  • Felsenstein KM, Goff SP. 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. [PMC free article] [PubMed]
  • Fleissner E, Tress E. Isolation of a ribonucleoprotein structure from oncornaviruses. J Virol. 1973 Dec;12(6):1612–1615. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Gauss P, Krassa KB, McPheeters DS, Nelson MA, 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. [PMC free article] [PubMed]
  • Giedroc DP, Keating KM, Williams KR, Coleman JE. The function of zinc in gene 32 protein from T4. Biochemistry. 1987 Aug 25;26(17):5251–5259. [PubMed]
  • Giedroc DP, Keating KM, Williams KR, Konigsberg WH, Coleman JE. 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Gorelick RJ, Henderson LE, Hanser JP, 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. [PMC free article] [PubMed]
  • Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. [PubMed]
  • Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. [PubMed]
  • Hsu HW, Schwartzberg P, Goff SP. Point mutations in the P30 domain of the gag gene of Moloney murine leukemia virus. Virology. 1985 Apr 15;142(1):211–214. [PubMed]
  • Karpel RL, Henderson LE, Oroszlan S. Interactions of retroviral structural proteins with single-stranded nucleic acids. J Biol Chem. 1987 Apr 15;262(11):4961–4967. [PubMed]
  • Katz RA, Terry RW, Skalka AM. 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Khandjian EW, Méric C. A procedure for Northern blot analysis of native RNA. Anal Biochem. 1986 Nov 15;159(1):227–232. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Leis J, Baltimore D, Bishop JM, Coffin J, Fleissner E, Goff SP, Oroszlan S, Robinson H, Skalka AM, Temin HM, et al. Standardized and simplified nomenclature for proteins common to all retroviruses. J Virol. 1988 May;62(5):1808–1809. [PMC free article] [PubMed]
  • Linial M, Medeiros E, Hayward WS. An avian oncovirus mutant (SE 21Q1b) deficient in genomic RNA: biological and biochemical characterization. Cell. 1978 Dec;15(4):1371–1381. [PubMed]
  • Manly KF, Anderson GR, Stoler DL. Harvey sarcoma virus genome contains no extensive sequences unrelated to those of other retroviruses except ras. J Virol. 1988 Sep;62(9):3540–3543. [PMC free article] [PubMed]
  • Mann R, Mulligan RC, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. [PubMed]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Méric C, Gouilloud E, Spahr PF. Mutations in Rous sarcoma virus nucleocapsid protein p12 (NC): deletions of Cys-His boxes. J Virol. 1988 Sep;62(9):3328–3333. [PMC free article] [PubMed]
  • Méric C, Spahr PF. 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. [PMC free article] [PubMed]
  • Mount SM, Rubin GM. Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins. Mol Cell Biol. 1985 Jul;5(7):1630–1638. [PMC free article] [PubMed]
  • Murphy JE, Goff SP. 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. [PMC free article] [PubMed]
  • Nissen-Meyer J, Abraham AK. Specificity of RNA binding by the structural protein (p10) of Friend murine leukemia virus. J Mol Biol. 1980 Sep 5;142(1):19–28. [PubMed]
  • Prats AC, Sarih L, Gabus C, Litvak S, Keith G, Darlix JL. 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. [PMC free article] [PubMed]
  • Rein A, McClure MR, Rice NR, Luftig RB, Schultz AM. 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. [PMC free article] [PubMed]
  • Rowe WP, Pugh WE, Hartley JW. Plaque assay techniques for murine leukemia viruses. Virology. 1970 Dec;42(4):1136–1139. [PubMed]
  • Schiff LA, Nibert ML, Fields BN. 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. [PMC free article] [PubMed]
  • Schulein M, Burnette WN, August JT. Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids. J Virol. 1978 Apr;26(1):54–60. [PMC free article] [PubMed]
  • Schwartzberg P, Colicelli J, Gordon ML, Goff SP. Mutations in the gag gene of Moloney murine leukemia virus: effects on production of virions and reverse transcriptase. J Virol. 1984 Mar;49(3):918–924. [PMC free article] [PubMed]
  • Scolnick EM, Vass WC, Howk RS, Duesberg PH. Defective retrovirus-like 30S RNA species of rat and mouse cells are infectious if packaged by type C helper virus. J Virol. 1979 Mar;29(3):964–972. [PMC free article] [PubMed]
  • Shields A, Witte WN, Rothenberg E, Baltimore D. High frequency of aberrant expression of Moloney murine leukemia virus in clonal infections. Cell. 1978 Jul;14(3):601–609. [PubMed]
  • Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed]
  • Topp WC. Normal rat cell lines deficient in nuclear thymidine kinase. Virology. 1981 Aug;113(1):408–411. [PubMed]
  • Wigler M, Sweet R, Sim GK, Wold B, Pellicer A, Lacy E, Maniatis T, Silverstein S, Axel R. Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell. 1979 Apr;16(4):777–785. [PubMed]
  • Williams KR, LoPresti MB, Setoguchi M, Konigsberg WH. Amino acid sequence of the T4 DNA helix-destabilizing protein. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4614–4617. [PMC free article] [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]
  • Young HA, Gonda MA, De Feo D, Ellis RW, Nagashima K, Scolnick EM. Heteroduplex analysis of cloned rat endogenous replication-defective (30 S) retrovirus and Harvey murine sarcoma virus. Virology. 1980 Nov;107(1):89–99. [PubMed]
  • Zoller MJ, 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. [PMC free article] [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...