1887

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Volume 84, Issue 6

Mutational analysis of two zinc-finger motifs in the nucleocapsid protein of simian immunodeficiency virus mac239 Free

Abstract

To clarify the physiological function of two zinc-finger (ZF) motifs in the nucleocapsid (NC) protein of simian immunodeficiency virus (SIV), we constructed three mutant viruses with alterations in either or both motifs using a molecular clone of SIVmac (SIVmac239). An immunoblot analysis of the cell lysates transfected with DNA mutated in either the first (ZF1) or second (ZF2) motif showed that the amount of partially processed Gag products (Pr46) was greater than that produced by the wild-type (WT). The genomic RNA contents in the viral particles released from the transfected cells were measured by quantitative RT-PCR. Values for the ZF1 and ZF2 mutants and the double mutant were 26, 20 and 7 % that of the WT, respectively, indicating that the two ZF motifs of SIVmac239 NC protein function almost equivalently with respect to RNA encapsidation and processing of Gag precursors. Despite the presence of some genomic RNA in the mutant viruses, they lost all viral infectivity. To determine the reason for this, we examined (using PCR) to which step viral DNA synthesis proceeded in the mutant viruses. We did not see any block up to the step of minus-strand DNA synthesis. However, plus-strand DNA synthesis after plus-strand transfer did not occur in any of the mutant viruses. These findings indicated that the mutations in the ZF motifs of SIVmac led to a loss of infectivity due partly to impairment of DNA synthesis, in addition to inefficient encapsidation of genomic RNA.

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2003-06-01
2024-04-24
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References

  1. Akahata W., Ido E., Shimada T., Katsuyama K., Yamamoto H., Uesaka H., Ui M., Kuwata T., Takahashi H., Hayami M. 2000; DNA vaccination of macaques by a full genome HIV-1 plasmid which produces noninfectious virus particles. Virology 275:116–124
     [Google Scholar]
  2. Aldovini A., Young R. A. 1990; Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol 64:1920–1926
     [Google Scholar]
  3. Allain B., Lapadat-Tapolsky M., Berlioz C., Darlix J. L. 1994; Transactivation of the minus-strand DNA transfer by nucleocapsid protein during reverse transcription of the retroviral genome. EMBO J 13:973–981
     [Google Scholar]
  4. Barat C., Lullien V., Schatz O., Keith G., Nugeyre M. T., Gruninger-Leitch F., Barre-Sinoussi F., LeGrice S. F., Darlix J. L. 1989; HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J 8:3279–3285
     [Google Scholar]
  5. Barat C., Schatz O., LeGrice S., Darlix J. L. 1993; Analysis of the interactions of HIV1 replication primer tRNALys,3 with nucleocapsid protein and reverse transcriptase. J Mol Biol 231:185–190
     [Google Scholar]
  6. Berg J. M. 1986; Potential metal-binding domains in nucleic acid binding proteins. Science 232:485–487
     [Google Scholar]
  7. Berkowitz R. D., Luban J., Goff S. P. 1993; Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. J Virol 67:7190–7200
     [Google Scholar]
  8. Berthoux L., Pechoux C., Ottmann M., Morel G., Darlix J. L. 1997; Mutations in the N-terminal domain of human immunodeficiency virus type 1 nucleocapsid protein affect virion core structure and proviral DNA synthesis. J Virol 71:6973–6981
     [Google Scholar]
  9. Covey S. N. 1986; Amino acid sequence homology in gag region of reverse transcribing elements and the coat protein gene of cauliflower mosaic virus. Nucleic Acids Res 14:623–633
     [Google Scholar]
  10. Dannull J., Surovoy A., Jung G., Moelling K. 1994; Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues. EMBO J 13:1525–1533
     [Google Scholar]
  11. Darlix J. L., Lapadat-Tapolsky M., De Rocquigny H., Roques B. P. 1995; First glimpses at structure–function relationships of the nucleocapsid protein of retroviruses. J Mol Biol 254:523–537
     [Google Scholar]
  12. De Rocquigny H., Gabus C., Vincent A., Fournie-Zaluski M. C., Roques B., Darlix J. L. 1992; Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers. Proc Natl Acad Sci U S A 89:6472–6476
     [Google Scholar]
  13. Dorfman T., Luban J., Goff S. P., Haseltine W. A., Gottlinger H. G. 1993; Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein. J Virol 67:6159–6169
     [Google Scholar]
  14. Garzino-Demo A., Gallo R. C., Arya S. K. 1995; Human immunodeficiency virus type 2 (HIV-2): packaging signal and associated negative regulatory element. Hum Gene Ther 6:177–184
     [Google Scholar]
  15. Gonsky J., Bacharach E., Goff S. P. 2001; Identification of residues of the Moloney murine leukemia virus nucleocapsid critical for viral DNA synthesis in vivo. J Virol 75:2616–2626
     [Google Scholar]
  16. Gorelick R. J., Henderson L. E., Hanser J. P., Rein A. 1988; 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 85:8420–8424
     [Google Scholar]
  17. Gorelick R. J., Nigida S. M. Jr, Bess J. W. Jr, Arthur L. O., Henderson L. E., Rein A. 1990; Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA. J Virol 64:3207–3211
     [Google Scholar]
  18. Gorelick R. J., Chabot D. J., Rein A., Henderson L. E., Arthur L. O. 1993; The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent. J Virol 67:4027–4036
     [Google Scholar]
  19. Gorelick R. J., Chabot D. J., Ott D. E., Gagliardi T. D., Rein A., Henderson L. E., Arthur L. O. 1996; Genetic analysis of the zinc finger in the Moloney murine leukemia virus nucleocapsid domain: replacement of zinc-coordinating residues with other zinc-coordinating residues yields noninfectious particles containing genomic RNA. J Virol 70:2593–2597
     [Google Scholar]
  20. Gorelick R. J., Benveniste R. E., Gagliardi T. D. 8 other authors 1999; Nucleocapsid protein zinc-finger mutants of simian immunodeficiency virus strain mne produce virions that are replication defective in vitro and in vivo. Virology 253:259–270
     [Google Scholar]
  21. Guo J., Henderson L. E., Bess J., Kane B., Levin J. G. 1997; Human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral DNA synthesis by inhibiting TAR-dependent self-priming from minus-strand strong-stop DNA. J Virol 71:5178–5188
     [Google Scholar]
  22. Guo J., Wu T., Anderson J., Kane B. F., Johnson D. G., Gorelick R. J., Henderson L. E., Levin J. G. 2000; Zinc finger structures in the human immunodeficiency virus type 1 nucleocapsid protein facilitate efficient minus- and plus-strand transfer. J Virol 74:8980–8988
     [Google Scholar]
  23. Kaye J. F., Lever A. M. 1999; Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation. J Virol 73:3023–3031
     [Google Scholar]
  24. Kestler H., Kodama T., Ringler D. 8 other authors 1990; Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus. Science 248:1109–1112
     [Google Scholar]
  25. Komatsu H., Tsukahara T., Tozawa H. 1996; Viral RNA binding properties of human immunodeficiency virus type-2 (HIV-2) nucleocapsid protein-derived synthetic peptides. Biochem Mol Biol Int 38:1143–1154
     [Google Scholar]
  26. Kuwata T., Igarashi T., Ido E., Jin M., Mizuno A., Chen J., Hayami M. 1995; Construction of human immunodeficiency virus 1/simian immunodeficiency virus strain mac chimeric viruses having vpr and/or nef of different parental origins and their in vitro and in vivo replication. J Gen Virol 76:2181–2191
     [Google Scholar]
  27. Maurer B., Bannert H., Darai G., Flugel R. M. 1988; Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus. J Virol 62:1590–1597
     [Google Scholar]
  28. Meric C., Goff S. P. 1989; Characterization of Moloney murine leukemia virus mutants with single-amino-acid substitutions in the Cys–His box of the nucleocapsid protein. J Virol 63:1558–1568
     [Google Scholar]
  29. Meric C., Darlix J. L., Spahr P. F. 1984; It is Rous sarcoma virus protein P12 and not P19 that binds tightly to Rous sarcoma virus RNA. J Mol Biol 173:531–538
     [Google Scholar]
  30. Meric C., Gouilloud E., Spahr P. F. 1988; Mutation in Rous sarcoma virus nucleocapsid protein P12NC: deletions of Cys–His boxes. J Virol 62:3328–3333
     [Google Scholar]
  31. Mizuno A., Ido E., Goto T., Kuwata T., Nakai M., Hayami M. 1996; Mutational analysis of two zinc finger motifs in HIV type 1 nucleocapsid proteins: effects on proteolytic processing of Gag precursors and particle formation. AIDS Res Hum Retrovir 12:793–800
     [Google Scholar]
  32. Naidu Y. M., Kestler H. W. D., Li Y. 8 other authors 1988; Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac. J Virol 62:4691–4696
     [Google Scholar]
  33. Prats A. C., Sarih L., Gabus C., Litvak S., Keith G., Darlix J. L. 1988; Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA. EMBO J 7:1777–1783
     [Google Scholar]
  34. Prats A. C., Roy C., Wang P. A., Erard M., Housset V., Gabus C., Paoletti C., Darlix J. L. 1990; Cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA. J Virol 64:774–783
     [Google Scholar]
  35. Prats A. C., Housset V., De Billy G., Cornille F., Prats H., Roques B., Darlix J. L. 1991; Viral RNA annealing activities of the nucleocapsid protein of Moloney murine leukemia virus are zinc independent. Nucleic Acids Res 19:3533–3541
     [Google Scholar]
  36. Shibata R., Kawamura M., Sakai H., Hayami M., Ishimoto A., Adachi A. 1991; Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells. J Virol 65:3514–3520
     [Google Scholar]
  37. Stewart L., Schatz G., Vogt V. M. 1990; Properties of avian retrovirus particles defective in viral protease. J Virol 64:5076–5092
     [Google Scholar]
  38. Suryanarayana K., Wiltrout T. A., Vasquez G. M., Hirsch V. M., Lifson J. D. 1998; Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction. AIDS Res Hum Retrovir 14:183–189
     [Google Scholar]
  39. Tanchou V., Decimo D., Pechoux C., Lener D., Rogemond V., Berthoux L., Ottmann M., Darlix J. L. 1998; Role of the N-terminal zinc finger of human immunodeficiency virus type 1 nucleocapsid protein in virus structure and replication. J Virol 72:4442–4447
     [Google Scholar]
  40. Urbaneja M. A., McGrath C. F., Kane B. P., Henderson L. E., Casas-Finet J. R. 2000; Nucleic acid binding properties of the simian immunodeficiency virus nucleocapsid protein NCp8. J Biol Chem 275:10394–10404
     [Google Scholar]
  41. Willey R. L., Smith D. H., Lasky L. A., Theodorer T. S., Earl P. L., Mos B., Capon D. L., Martin M. A. 1988; In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J Virol 62:139–147
     [Google Scholar]
  42. Wu T., Guo J., Bess J., Henderson L. E., Levin J. G. 1999; Molecular requirements for human immunodeficiency virus type 1 plus-strand transfer: analysis in reconstituted and endogenous reverse transcription systems. J Virol 73:4794–4805
     [Google Scholar]
  43. Xiao Y., Kuwata T., Miura T., Hayami M., Shida H. 2000; Dox-dependent SIVmac with tetracycline-inducible promoter in the U3 promoter region. Virology 269:268–275
     [Google Scholar]
  44. You J. C., McHenry C. S. 1994; Human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription. J Biol Chem 269:31491–31495
     [Google Scholar]
  45. Yovandich J. L., Chertova E. N., Kane B. P., Gagliardi T. D., Bess J. W. Jr, Sowder R. C. II, Henderson L. E., Gorelick R. J. 2001; Alteration of zinc-binding residues of simian immunodeficiency virus p8NC results in subtle differences in gag processing and virion maturation associated with degradative loss of mutant NC. J Virol 75:115–124
     [Google Scholar]
  46. Yu Q., Darlix J. L. 1996; The zinc finger of nucleocapsid protein of Friend murine leukemia virus is critical for proviral DNA synthesis in vivo. J Virol 70:5791–5798
     [Google Scholar]
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Mutational analysis of two zinc-finger motifs in the nucleocapsid protein of simian immunodeficiency virus mac239
J. Gen. Virol. 84, 1641 (2003); https://doi.org/10.1099/vir.0.18865-0
/content/journal/jgv/10.1099/vir.0.18865-0
/content/journal/jgv/10.1099/vir.0.18865-0
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