Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
RNA. 2001 Apr; 7(4): 576–584.
doi: 10.1017/s1355838201002023
PMCID: PMC1370111
PMID: 11345436

Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection.

P Purohit, S Dupont, M Stevenson, and M R Green
Author information Copyright and License information Disclaimer

Abstract

The human immunodeficiency virus type-1 matrix protein (HIV-1 MA) is a multifunctional structural protein synthesized as part of the Pr55 gag polyprotein. We have used in vitro genetic selection to identify an RNA consensus sequence that specifically interacts with MA (Kd = 5 x 10(-7) M). This 13-nt MA binding consensus sequence bears a high degree of homology (77%) to a region (nt 1433-1446) within the POL open reading frame of the HIV-1 genome (consensus sequence from 38 HIV-1 strains). Chemical interference experiments identified the nucleotides within the MA binding consensus sequence involved in direct contact with MA. We further demonstrate that this RNA-protein interaction is mediated through a stretch of basic amino acids within MA. Mutations that disrupt the interaction between MA and its RNA binding site within the HIV-1 genome resulted in a measurable decrease in viral replication.

Full Text

The Full Text of this article is available as a PDF (645K).

Selected References

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

  • Aldovini A, Young RA. 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. [PMC free article] [PubMed] [Google Scholar]
  • Allen P, Collins B, Brown D, Hostomsky Z, Gold L. A specific RNA structural motif mediates high affinity binding by the HIV-1 nucleocapsid protein (NCp7). Virology. 1996 Nov 15;225(2):306–315. [PubMed] [Google Scholar]
  • Bartel DP, Zapp ML, Green MR, Szostak JW. HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA. Cell. 1991 Nov 1;67(3):529–536. [PubMed] [Google Scholar]
  • Berglund JA, Charpentier B, Rosbash M. A high affinity binding site for the HIV-1 nucleocapsid protein. Nucleic Acids Res. 1997 Mar 1;25(5):1042–1049. [PMC free article] [PubMed] [Google Scholar]
  • Berkowitz R, Fisher J, Goff SP. RNA packaging. Curr Top Microbiol Immunol. 1996;214:177–218. [PubMed] [Google Scholar]
  • Berkowitz RD, Luban J, Goff SP. Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. J Virol. 1993 Dec;67(12):7190–7200. [PMC free article] [PubMed] [Google Scholar]
  • Bowerman B, Brown PO, Bishop JM, Varmus HE. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. [PubMed] [Google Scholar]
  • Bukrinskaya AG, Vorkunova GK, Tentsov YYu HIV-1 matrix protein p17 resides in cell nuclei in association with genomic RNA. AIDS Res Hum Retroviruses. 1992 Oct;8(10):1795–1801. [PubMed] [Google Scholar]
  • Bukrinsky MI, Haggerty S, Dempsey MP, Sharova N, Adzhubel A, Spitz L, Lewis P, Goldfarb D, Emerman M, Stevenson M. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature. 1993 Oct 14;365(6447):666–669. [PMC free article] [PubMed] [Google Scholar]
  • Bukrinsky MI, Sharova N, McDonald TL, Pushkarskaya T, Tarpley WG, Stevenson M. Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6125–6129. [PMC free article] [PubMed] [Google Scholar]
  • Churcher MJ, Lamont C, Hamy F, Dingwall C, Green SM, Lowe AD, Butler JG, Gait MJ, Karn J. High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region. J Mol Biol. 1993 Mar 5;230(1):90–110. [PubMed] [Google Scholar]
  • Clavel F, Orenstein JM. A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology. J Virol. 1990 Oct;64(10):5230–5234. [PMC free article] [PubMed] [Google Scholar]
  • Darlix JL, Lapadat-Tapolsky M, de Rocquigny H, Roques BP. First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. J Mol Biol. 1995 Dec 8;254(4):523–537. [PubMed] [Google Scholar]
  • Dupont S, Sharova N, DéHoratius C, Virbasius CM, Zhu X, Bukrinskaya AG, Stevenson M, Green MR. A novel nuclear export activity in HIV-1 matrix protein required for viral replication. Nature. 1999 Dec 9;402(6762):681–685. [PubMed] [Google Scholar]
  • Farnet CM, Haseltine WA. Circularization of human immunodeficiency virus type 1 DNA in vitro. J Virol. 1991 Dec;65(12):6942–6952. [PMC free article] [PubMed] [Google Scholar]
  • Hahn BH, Shaw GM, Arya SK, Popovic M, Gallo RC, Wong-Staal F. Molecular cloning and characterization of the HTLV-III virus associated with AIDS. Nature. 1984 Nov 8;312(5990):166–169. [PubMed] [Google Scholar]
  • Heinzinger NK, Bukrinsky MI, Haggerty SA, Ragland AM, Kewalramani V, Lee MA, Gendelman HE, Ratner L, Stevenson M, Emerman M. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7311–7315. [PMC free article] [PubMed] [Google Scholar]
  • Horwitz BH, DiMaio D. Saturation mutagenesis using mixed oligonucleotides and M13 templates containing uracil. Methods Enzymol. 1990;185:599–611. [PubMed] [Google Scholar]
  • Hüttenhofer A, Noller HF. Footprinting mRNA-ribosome complexes with chemical probes. EMBO J. 1994 Aug 15;13(16):3892–3901. [PMC free article] [PubMed] [Google Scholar]
  • Kiernan RE, Freed EO. Cleavage of the murine leukemia virus transmembrane env protein by human immunodeficiency virus type 1 protease: transdominant inhibition by matrix mutations. J Virol. 1998 Dec;72(12):9621–9627. [PMC free article] [PubMed] [Google Scholar]
  • Kiernan RE, Ono A, Englund G, Freed EO. Role of matrix in an early postentry step in the human immunodeficiency virus type 1 life cycle. J Virol. 1998 May;72(5):4116–4126. [PMC free article] [PubMed] [Google Scholar]
  • Kjems J, Calnan BJ, Frankel AD, Sharp PA. Specific binding of a basic peptide from HIV-1 Rev. EMBO J. 1992 Mar;11(3):1119–1129. [PMC free article] [PubMed] [Google Scholar]
  • Kondo E, Mammano F, Cohen EA, Göttlinger HG. The p6gag domain of human immunodeficiency virus type 1 is sufficient for the incorporation of Vpr into heterologous viral particles. J Virol. 1995 May;69(5):2759–2764. [PMC free article] [PubMed] [Google Scholar]
  • Lavallée C, Yao XJ, Ladha A, Göttlinger H, Haseltine WA, Cohen EA. Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles. J Virol. 1994 Mar;68(3):1926–1934. [PMC free article] [PubMed] [Google Scholar]
  • Lochrie MA, Waugh S, Pratt DG, Jr, Clever J, Parslow TG, Polisky B. In vitro selection of RNAs that bind to the human immunodeficiency virus type-1 gag polyprotein. Nucleic Acids Res. 1997 Jul 15;25(14):2902–2910. [PMC free article] [PubMed] [Google Scholar]
  • Lu YL, Spearman P, Ratner L. Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions. J Virol. 1993 Nov;67(11):6542–6550. [PMC free article] [PubMed] [Google Scholar]
  • Luban J, Goff SP. Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein. J Virol. 1991 Jun;65(6):3203–3212. [PMC free article] [PubMed] [Google Scholar]
  • Luban J, Goff SP. Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J Virol. 1994 Jun;68(6):3784–3793. [PMC free article] [PubMed] [Google Scholar]
  • Miller MD, Farnet CM, Bushman FD. Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. J Virol. 1997 Jul;71(7):5382–5390. [PMC free article] [PubMed] [Google Scholar]
  • Mirfakhrai M, Weiner AM. Chemical Cleveland mapping: a rapid technique for characterization of crosslinked nucleic acid-protein complexes. Nucleic Acids Res. 1993 Jul 25;21(15):3591–3592. [PMC free article] [PubMed] [Google Scholar]
  • Paxton W, Connor RI, Landau NR. Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol. 1993 Dec;67(12):7229–7237. [PMC free article] [PubMed] [Google Scholar]
  • Purohit P, Stern S. Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit. Nature. 1994 Aug 25;370(6491):659–662. [PubMed] [Google Scholar]
  • Sakaguchi K, Zambrano N, Baldwin ET, Shapiro BA, Erickson JW, Omichinski JG, Clore GM, Gronenborn AM, Appella E. Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5219–5223. [PMC free article] [PubMed] [Google Scholar]
  • Schneider DJ, Feigon J, Hostomsky Z, Gold L. High-affinity ssDNA inhibitors of the reverse transcriptase of type 1 human immunodeficiency virus. Biochemistry. 1995 Jul 25;34(29):9599–9610. [PubMed] [Google Scholar]
  • Singh R, Valcárcel J, Green MR. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science. 1995 May 26;268(5214):1173–1176. [PubMed] [Google Scholar]
  • Tao J, Frankel AD. Arginine-binding RNAs resembling TAR identified by in vitro selection. Biochemistry. 1996 Feb 20;35(7):2229–2238. [PubMed] [Google Scholar]
  • Tuerk C, MacDougal-Waugh S. In vitro evolution of functional nucleic acids: high-affinity RNA ligands of HIV-1 proteins. Gene. 1993 Dec 27;137(1):33–39. [PubMed] [Google Scholar]
  • Tuerk C, MacDougal S, Gold L. RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6988–6992. [PMC free article] [PubMed] [Google Scholar]
  • Valcárcel J, Singh R, Zamore PD, Green MR. The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature. 1993 Mar 11;362(6416):171–175. [PubMed] [Google Scholar]
  • von Schwedler U, Kornbluth RS, Trono D. The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6992–6996. [PMC free article] [PubMed] [Google Scholar]
  • Wyatt JR, Vickers TA, Roberson JL, Buckheit RW, Jr, Klimkait T, DeBaets E, Davis PW, Rayner B, Imbach JL, Ecker DJ. Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virus envelope-mediated cell fusion. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1356–1360. [PMC free article] [PubMed] [Google Scholar]
  • Zapp ML, Stern S, Green MR. Small molecules that selectively block RNA binding of HIV-1 Rev protein inhibit Rev function and viral production. Cell. 1993 Sep 24;74(6):969–978. [PubMed] [Google Scholar]
Articles from RNA are provided here courtesy of The RNA Society