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. 1996 Aug;70(8):4996–5004. doi: 10.1128/jvi.70.8.4996-5004.1996

Human immunodeficiency virus Type 1 nucleocapsid protein (NCp7) directs specific initiation of minus-strand DNA synthesis primed by human tRNA(Lys3) in vitro: studies of viral RNA molecules mutated in regions that flank the primer binding site.

X Li 1, Y Quan 1, E J Arts 1, Z Li 1, B D Preston 1, H de Rocquigny 1, B P Roques 1, J L Darlix 1, L Kleiman 1, M A Parniak 1, M A Wainberg 1
PMCID: PMC190453  PMID: 8764006

Abstract

Retroviral reverse transcription starts near the 5' end of unspliced viral RNA at a sequence called the primer binding site (PBS), where the tRNA primer anneals to the RNA template for initiation of DNA synthesis. We have investigated the roles of NCp7 in annealing of primer tRNA(Lys3) to the PBS and in reverse transcriptase (RT) activity, using a cell-free reverse transcription reaction mixture consisting of various 5' viral RNA templates, natural primer tRNA(Lys3) or synthetic primer, human immunodeficiency virus type I (HIV-1) nucleocapsid protein (NCp7), and HIV-1 RT. In the presence of tRNA(Lys3), NCp7 was found to stimulate synthesis of minus-strand strong-stop DNA [(-)ssDNA], consistent with previous reports. However, specific DNA synthesis was observed only at a NCp7/RNA ratio similar to that predicted to be present in virions. Moreover, at these concentrations, NCp7 inhibited the synthesis of nonspecific reverse-transcribed DNA products, which are initiated because of self-priming by RNA templates. In contrast to results obtained with tRNA(Lys3) as primer, NCp7 inhibited the synthesis of (-)ssDNA products primed by an 18-nucleotide (nt) ribonucleotide (rPR), complementary to the PBS, even though rPR can initiate synthesis of such material in the absence of preannealing with NCp7. Primer placement band shift assays showed that NCp7 was necessary for efficient formation of the tRNA-RNA complex. In contrast, NCp7 was found to prevent formation of the rPR-RNA complex. Since NCp7 appears to exert opposite effects (annealing versus dissociation) on tRNA(Lys3) and rPR substrates, the non-PBS binding regions of the tRNA(Lys3) molecule may play a role in the annealing of tRNA to the template. We also investigated the roles of an A-rich loop upstream of the PBS, a 7-nt region immediately downstream of the PBS, and a 54-nt deletion further downstream of the PBS in interactions with tRNA(Lys3). We found that deletions in the 54-nt region that may prevent formation of the U5-leader stem prevented tRNA(Lys3) placement and priming, while deletions in the A-rich loop or the 7-nt sequence had relatively minor effects in this regard.

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Selected References

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  1. Aiyar A., Cobrinik D., Ge Z., Kung H. J., Leis J. Interaction between retroviral U5 RNA and the T psi C loop of the tRNA(Trp) primer is required for efficient initiation of reverse transcription. J Virol. 1992 Apr;66(4):2464–2472. doi: 10.1128/jvi.66.4.2464-2472.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiyar A., Ge Z., Leis J. A specific orientation of RNA secondary structures is required for initiation of reverse transcription. J Virol. 1994 Feb;68(2):611–618. doi: 10.1128/jvi.68.2.611-618.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Allain B., Lapadat-Tapolsky M., Berlioz C., Darlix J. L. Transactivation of the minus-strand DNA transfer by nucleocapsid protein during reverse transcription of the retroviral genome. EMBO J. 1994 Feb 15;13(4):973–981. doi: 10.1002/j.1460-2075.1994.tb06342.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arts E. J., Li X., Gu Z., Kleiman L., Parniak M. A., Wainberg M. A. Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction. J Biol Chem. 1994 May 20;269(20):14672–14680. [PubMed] [Google Scholar]
  5. Barat C., Lullien V., Schatz O., Keith G., Nugeyre M. T., Grüninger-Leitch F., Barré-Sinoussi F., LeGrice S. F., Darlix J. L. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J. 1989 Nov;8(11):3279–3285. doi: 10.1002/j.1460-2075.1989.tb08488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barat C., Schatz O., Le Grice S., Darlix J. L. Analysis of the interactions of HIV1 replication primer tRNA(Lys,3) with nucleocapsid protein and reverse transcriptase. J Mol Biol. 1993 May 20;231(2):185–190. doi: 10.1006/jmbi.1993.1273. [DOI] [PubMed] [Google Scholar]
  7. Baudin F., Marquet R., Isel C., Darlix J. L., Ehresmann B., Ehresmann C. Functional sites in the 5' region of human immunodeficiency virus type 1 RNA form defined structural domains. J Mol Biol. 1993 Jan 20;229(2):382–397. doi: 10.1006/jmbi.1993.1041. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Berkhout B., Schoneveld I. Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site. Nucleic Acids Res. 1993 Mar 11;21(5):1171–1178. doi: 10.1093/nar/21.5.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bess J. W., Jr, Powell P. J., Issaq H. J., Schumack L. J., Grimes M. K., Henderson L. E., Arthur L. O. Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses. J Virol. 1992 Feb;66(2):840–847. doi: 10.1128/jvi.66.2.840-847.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Blain S. W., Hendrickson W. A., Goff S. P. Reversion of a Moloney murine leukemia virus RNase H mutant at a second site restores enzyme function and infectivity. J Virol. 1995 Aug;69(8):5113–5116. doi: 10.1128/jvi.69.8.5113-5116.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cobrinik D., Aiyar A., Ge Z., Katzman M., Huang H., Leis J. Overlapping retrovirus U5 sequence elements are required for efficient integration and initiation of reverse transcription. J Virol. 1991 Jul;65(7):3864–3872. doi: 10.1128/jvi.65.7.3864-3872.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cobrinik D., Soskey L., Leis J. A retroviral RNA secondary structure required for efficient initiation of reverse transcription. J Virol. 1988 Oct;62(10):3622–3630. doi: 10.1128/jvi.62.10.3622-3630.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Coffin J. M. Structure, replication, and recombination of retrovirus genomes: some unifying hypotheses. J Gen Virol. 1979 Jan;42(1):1–26. doi: 10.1099/0022-1317-42-1-1. [DOI] [PubMed] [Google Scholar]
  15. Darlix J. L., Lapadat-Tapolsky M., de Rocquigny H., Roques B. P. First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. J Mol Biol. 1995 Dec 8;254(4):523–537. doi: 10.1006/jmbi.1995.0635. [DOI] [PubMed] [Google Scholar]
  16. Darlix J. L., Schwager M., Spahr P. F., Bromley P. A. Analysis of the secondary and tertiary structures of Rous sarcoma virus RNA. Nucleic Acids Res. 1980 Aug 11;8(15):3335–3354. doi: 10.1093/nar/8.15.3335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Darlix J. L., Vincent A., Gabus C., de Rocquigny H., Roques B. Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA. C R Acad Sci III. 1993 Aug;316(8):763–771. [PubMed] [Google Scholar]
  18. Das A. T., Klaver B., Berkhout B. Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys). J Virol. 1995 May;69(5):3090–3097. doi: 10.1128/jvi.69.5.3090-3097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. De Rocquigny H., Gabus C., Vincent A., Fournié-Zaluski M. C., Roques B., Darlix J. L. 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. 1992 Jul 15;89(14):6472–6476. doi: 10.1073/pnas.89.14.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fitzgerald D. W., Coleman J. E. Physicochemical properties of cloned nucleocapsid protein from HIV. Interactions with metal ions. Biochemistry. 1991 May 28;30(21):5195–5201. doi: 10.1021/bi00235a012. [DOI] [PubMed] [Google Scholar]
  21. Harrison G. P., Lever A. M. The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure. J Virol. 1992 Jul;66(7):4144–4153. doi: 10.1128/jvi.66.7.4144-4153.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Herschlag D., Khosla M., Tsuchihashi Z., Karpel R. L. An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis. EMBO J. 1994 Jun 15;13(12):2913–2924. doi: 10.1002/j.1460-2075.1994.tb06586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Huber H. E., McCoy J. M., Seehra J. S., Richardson C. C. Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. J Biol Chem. 1989 Mar 15;264(8):4669–4678. [PubMed] [Google Scholar]
  24. Isel C., Ehresmann C., Keith G., Ehresmann B., Marquet R. Initiation of reverse transcription of HIV-1: secondary structure of the HIV-1 RNA/tRNA(3Lys) (template/primer). J Mol Biol. 1995 Mar 24;247(2):236–250. doi: 10.1006/jmbi.1994.0136. [DOI] [PubMed] [Google Scholar]
  25. Ji X., Klarmann G. J., Preston B. D. Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro. Biochemistry. 1996 Jan 9;35(1):132–143. doi: 10.1021/bi951707e. [DOI] [PubMed] [Google Scholar]
  26. Jiang M., Mak J., Ladha A., Cohen E., Klein M., Rovinski B., Kleiman L. Identification of tRNAs incorporated into wild-type and mutant human immunodeficiency virus type 1. J Virol. 1993 Jun;67(6):3246–3253. doi: 10.1128/jvi.67.6.3246-3253.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Junghans R. P., Boone L. R., Skalka A. M. Retroviral DNA H structures: displacement-assimilation model of recombination. Cell. 1982 Aug;30(1):53–62. doi: 10.1016/0092-8674(82)90011-3. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Khan R., Giedroc D. P. Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA. J Biol Chem. 1992 Apr 5;267(10):6689–6695. [PubMed] [Google Scholar]
  30. Klarmann G. J., Schauber C. A., Preston B. D. Template-directed pausing of DNA synthesis by HIV-1 reverse transcriptase during polymerization of HIV-1 sequences in vitro. J Biol Chem. 1993 May 5;268(13):9793–9802. [PubMed] [Google Scholar]
  31. Lapadat-Tapolsky M., Pernelle C., Borie C., Darlix J. L. Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1. Nucleic Acids Res. 1995 Jul 11;23(13):2434–2441. doi: 10.1093/nar/23.13.2434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Li X., Mak J., Arts E. J., Gu Z., Kleiman L., Wainberg M. A., Parniak M. A. Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication. J Virol. 1994 Oct;68(10):6198–6206. doi: 10.1128/jvi.68.10.6198-6206.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Litvak S., Sarih-Cottin L., Fournier M., Andreola M., Tarrago-Litvak L. Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase. Trends Biochem Sci. 1994 Mar;19(3):114–118. doi: 10.1016/0968-0004(94)90203-8. [DOI] [PubMed] [Google Scholar]
  34. Morellet N., Jullian N., De Rocquigny H., Maigret B., Darlix J. L., Roques B. P. Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR. EMBO J. 1992 Aug;11(8):3059–3065. doi: 10.1002/j.1460-2075.1992.tb05377.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Peliska J. A., Balasubramanian S., Giedroc D. P., Benkovic S. J. Recombinant HIV-1 nucleocapsid protein accelerates HIV-1 reverse transcriptase catalyzed DNA strand transfer reactions and modulates RNase H activity. Biochemistry. 1994 Nov 22;33(46):13817–13823. doi: 10.1021/bi00250a036. [DOI] [PubMed] [Google Scholar]
  36. Picard V., Ersdal-Badju E., Lu A., Bock S. C. A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res. 1994 Jul 11;22(13):2587–2591. doi: 10.1093/nar/22.13.2587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  39. Rhim H., Park J., Morrow C. D. Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription. J Virol. 1991 Sep;65(9):4555–4564. doi: 10.1128/jvi.65.9.4555-4564.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rodríguez-Rodríguez L., Tsuchihashi Z., Fuentes G. M., Bambara R. A., Fay P. J. Influence of human immunodeficiency virus nucleocapsid protein on synthesis and strand transfer by the reverse transcriptase in vitro. J Biol Chem. 1995 Jun 23;270(25):15005–15011. doi: 10.1074/jbc.270.25.15005. [DOI] [PubMed] [Google Scholar]
  41. South T. L., Blake P. R., Sowder R. C., 3rd, Arthur L. O., Henderson L. E., Summers M. F. The nucleocapsid protein isolated from HIV-1 particles binds zinc and forms retroviral-type zinc fingers. Biochemistry. 1990 Aug 28;29(34):7786–7789. doi: 10.1021/bi00486a002. [DOI] [PubMed] [Google Scholar]
  42. Surovoy A., Dannull J., Moelling K., Jung G. Conformational and nucleic acid binding studies on the synthetic nucleocapsid protein of HIV-1. J Mol Biol. 1993 Jan 5;229(1):94–104. doi: 10.1006/jmbi.1993.1011. [DOI] [PubMed] [Google Scholar]
  43. Tanchou V., Gabus C., Rogemond V., Darlix J. L. Formation of stable and functional HIV-1 nucleoprotein complexes in vitro. J Mol Biol. 1995 Oct 6;252(5):563–571. doi: 10.1006/jmbi.1995.0520. [DOI] [PubMed] [Google Scholar]
  44. Temin H. M. Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus genetic variation. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6900–6903. doi: 10.1073/pnas.90.15.6900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tsuchihashi Z., Brown P. O. DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein. J Virol. 1994 Sep;68(9):5863–5870. doi: 10.1128/jvi.68.9.5863-5870.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wakefield J. K., Wolf A. G., Morrow C. D. Human immunodeficiency virus type 1 can use different tRNAs as primers for reverse transcription but selectively maintains a primer binding site complementary to tRNA(3Lys). J Virol. 1995 Oct;69(10):6021–6029. doi: 10.1128/jvi.69.10.6021-6029.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weiss S., König B., Morikawa Y., Jones I. Recombinant HIV-1 nucleocapsid protein p15 produced as a fusion protein with glutathione S-transferase in Escherichia coli mediates dimerization and enhances reverse transcription of retroviral RNA. Gene. 1992 Nov 16;121(2):203–212. doi: 10.1016/0378-1119(92)90123-7. [DOI] [PubMed] [Google Scholar]
  48. Whitcomb J. M., Ortiz-Conde B. A., Hughes S. H. Replication of avian leukosis viruses with mutations at the primer binding site: use of alternative tRNAs as primers. J Virol. 1995 Oct;69(10):6228–6238. doi: 10.1128/jvi.69.10.6228-6238.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wöhrl B. M., Ehresmann B., Keith G., Le Grice S. F. Nuclease footprinting of human immunodeficiency virus reverse transcriptase/tRNA(Lys-3) complexes. J Biol Chem. 1993 Jun 25;268(18):13617–13624. [PubMed] [Google Scholar]
  50. You J. C., McHenry C. S. Human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription. J Biol Chem. 1994 Dec 16;269(50):31491–31495. [PubMed] [Google Scholar]
  51. de Rocquigny H., Ficheux D., Gabus C., Fournié-Zaluski M. C., Darlix J. L., Roques B. P. First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form. Biochem Biophys Res Commun. 1991 Oct 31;180(2):1010–1018. doi: 10.1016/s0006-291x(05)81166-0. [DOI] [PubMed] [Google Scholar]

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