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. 1997 Dec;71(12):9259–9269. doi: 10.1128/jvi.71.12.9259-9269.1997

Discontinuous plus-strand DNA synthesis in human immunodeficiency virus type 1-infected cells and in a partially reconstituted cell-free system.

G J Klarmann 1, H Yu 1, X Chen 1, J P Dougherty 1, B D Preston 1
PMCID: PMC230228  PMID: 9371584

Abstract

Human immunodeficiency virus type 1 (HIV-1) replication requires conversion of viral RNA to double-stranded DNA. To better understand the molecular mechanisms of this process, we examined viral DNA synthesis in a simple cell-free system that uses the activities of HIV-1 reverse transcriptase to convert regions of single-stranded HIV-1 RNA to double-stranded DNA in a single incubation. This system recapitulated several of the required intermediate steps of viral DNA synthesis: RNA-templated minus-strand polymerization, preferential plus-strand initiation at the central and 3' HIV-1 polypurine tracts, and DNA-templated plus-strand polymerization. Secondary sites of plus-strand initiation were also observed at low frequency both in the cell-free system and in cultured virus. Direct comparison of viral and cell-free products revealed differences in the precision and selectivity of plus-strand initiation, suggesting that the cell-free system lacks one or more essential replication components. These studies provide clues about mechanisms of plus-strand initiation and serve as a starting point for the development of more complex multicomponent cell-free systems.

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Selected References

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  1. Abbotts J., Bebenek K., Kunkel T. A., Wilson S. H. Mechanism of HIV-1 reverse transcriptase. Termination of processive synthesis on a natural DNA template is influenced by the sequence of the template-primer stem. J Biol Chem. 1993 May 15;268(14):10312–10323. [PubMed] [Google Scholar]
  2. Arts E. J., Wainberg M. A. Human immunodeficiency virus type 1 reverse transcriptase and early events in reverse transcription. Adv Virus Res. 1996;46:97–163. doi: 10.1016/s0065-3527(08)60071-8. [DOI] [PubMed] [Google Scholar]
  3. Bandyopadhyay A. K. Effect of Rauscher leukemia virus-specific proteins on reverse transcriptase. Binding between reverse transcriptase and p30. J Biol Chem. 1977 Aug 25;252(16):5883–5887. [PubMed] [Google Scholar]
  4. Barbosa P., Charneau P., Dumey N., Clavel F. Kinetic analysis of HIV-1 early replicative steps in a coculture system. AIDS Res Hum Retroviruses. 1994 Jan;10(1):53–59. doi: 10.1089/aid.1994.10.53. [DOI] [PubMed] [Google Scholar]
  5. Borroto-Esoda K., Boone L. R. Equine infectious anemia virus and human immunodeficiency virus DNA synthesis in vitro: characterization of the endogenous reverse transcriptase reaction. J Virol. 1991 Apr;65(4):1952–1959. doi: 10.1128/jvi.65.4.1952-1959.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bowerman B., Brown P. O., Bishop J. M., Varmus H. E. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. doi: 10.1101/gad.3.4.469. [DOI] [PubMed] [Google Scholar]
  7. Bukrinsky M. I., Sharova N., McDonald T. L., Pushkarskaya T., Tarpley W. G., 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. doi: 10.1073/pnas.90.13.6125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Charneau P., Alizon M., Clavel F. A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication. J Virol. 1992 May;66(5):2814–2820. doi: 10.1128/jvi.66.5.2814-2820.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Charneau P., Clavel F. A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract. J Virol. 1991 May;65(5):2415–2421. doi: 10.1128/jvi.65.5.2415-2421.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clark J. M., Joyce C. M., Beardsley G. P. Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli. J Mol Biol. 1987 Nov 5;198(1):123–127. doi: 10.1016/0022-2836(87)90462-1. [DOI] [PubMed] [Google Scholar]
  11. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fu T. B., Taylor J. When retroviral reverse transcriptases reach the end of their RNA templates. J Virol. 1992 Jul;66(7):4271–4278. doi: 10.1128/jvi.66.7.4271-4278.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuentes G. M., Rodríguez-Rodríguez L., Fay P. J., Bambara R. A. Use of an oligoribonucleotide containing the polypurine tract sequence as a primer by HIV reverse transcriptase. J Biol Chem. 1995 Nov 24;270(47):28169–28176. doi: 10.1074/jbc.270.47.28169. [DOI] [PubMed] [Google Scholar]
  14. Gallay P., Swingler S., Song J., Bushman F., Trono D. HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase. Cell. 1995 Nov 17;83(4):569–576. doi: 10.1016/0092-8674(95)90097-7. [DOI] [PubMed] [Google Scholar]
  15. Harris J. D., Scott J. V., Traynor B., Brahic M., Stowring L., Ventura P., Haase A. T., Peluso R. Visna virus DNA: discovery of a novel gapped structure. Virology. 1981 Sep;113(2):573–583. doi: 10.1016/0042-6822(81)90185-9. [DOI] [PubMed] [Google Scholar]
  16. Heyman T., Agoutin B., Friant S., Wilhelm F. X., Wilhelm M. L. Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 3' LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition. J Mol Biol. 1995 Oct 20;253(2):291–303. doi: 10.1006/jmbi.1995.0553. [DOI] [PubMed] [Google Scholar]
  17. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  18. Hu W. S., Temin H. M. Retroviral recombination and reverse transcription. Science. 1990 Nov 30;250(4985):1227–1233. doi: 10.1126/science.1700865. [DOI] [PubMed] [Google Scholar]
  19. Huber H. E., Richardson C. C. Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase. J Biol Chem. 1990 Jun 25;265(18):10565–10573. [PubMed] [Google Scholar]
  20. Hung P. P., Lee S. G. Isolation of nucleic acid-binding protein: stimulation of reverse transcriptase-catalysed DNA synthesis. Nature. 1976 Feb 12;259(5543):499–502. doi: 10.1038/259499a0. [DOI] [PubMed] [Google Scholar]
  21. Hungnes O., Tjotta E., Grinde B. The plus strand is discontinuous in a subpopulation of unintegrated HIV-1 DNA. Arch Virol. 1991;116(1-4):133–141. doi: 10.1007/BF01319237. [DOI] [PubMed] [Google Scholar]
  22. Hungnes O., Tjøtta E., Grinde B. Mutations in the central polypurine tract of HIV-1 result in delayed replication. Virology. 1992 Sep;190(1):440–442. doi: 10.1016/0042-6822(92)91230-r. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Kim S. Y., Byrn R., Groopman J., Baltimore D. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression. J Virol. 1989 Sep;63(9):3708–3713. doi: 10.1128/jvi.63.9.3708-3713.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kimpton J., Emerman M. Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol. 1992 Apr;66(4):2232–2239. doi: 10.1128/jvi.66.4.2232-2239.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Kulkosky J., Skalka A. M. Molecular mechanism of retroviral DNA integration. Pharmacol Ther. 1994;61(1-2):185–203. doi: 10.1016/0163-7258(94)90062-0. [DOI] [PubMed] [Google Scholar]
  29. Kupiec J. J., Tobaly-Tapiero J., Canivet M., Santillana-Hayat M., Flügel R. M., Périès J., Emanoil-Ravier R. Evidence for a gapped linear duplex DNA intermediate in the replicative cycle of human and simian spumaviruses. Nucleic Acids Res. 1988 Oct 25;16(20):9557–9565. doi: 10.1093/nar/16.20.9557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Larder B. A., Kemp S. D., Purifoy D. J. Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4803–4807. doi: 10.1073/pnas.86.13.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Leis J. P., Hurwitz J. Isolation and characterization of a protein that stimulates DNA synthesis from avian myeloblastosis virus. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2331–2335. doi: 10.1073/pnas.69.8.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lori F., di Marzo Veronese F., de Vico A. L., Lusso P., Reitz M. S., Jr, Gallo R. C. Viral DNA carried by human immunodeficiency virus type 1 virions. J Virol. 1992 Aug;66(8):5067–5074. doi: 10.1128/jvi.66.8.5067-5074.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mansky L. M., Temin H. M. Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. J Virol. 1995 Aug;69(8):5087–5094. doi: 10.1128/jvi.69.8.5087-5094.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miller M. D., Wang B., Bushman F. D. Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro. J Virol. 1995 Jun;69(6):3938–3944. doi: 10.1128/jvi.69.6.3938-3944.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mizrahi V., Usdin M. T., Harington A., Dudding L. R. Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase. Nucleic Acids Res. 1990 Sep 25;18(18):5359–5363. doi: 10.1093/nar/18.18.5359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Olsen J. C., Swanstrom R. A new pathway in the generation of defective retrovirus DNA. J Virol. 1985 Dec;56(3):779–789. doi: 10.1128/jvi.56.3.779-789.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Patel P. H., Preston B. D. Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):549–553. doi: 10.1073/pnas.91.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Powell M. D., Levin J. G. Sequence and structural determinants required for priming of plus-strand DNA synthesis by the human immunodeficiency virus type 1 polypurine tract. J Virol. 1996 Aug;70(8):5288–5296. doi: 10.1128/jvi.70.8.5288-5296.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Preston B. D., Dougherty J. P. Mechanisms of retroviral mutation. Trends Microbiol. 1996 Jan;4(1):16–21. doi: 10.1016/0966-842x(96)81500-9. [DOI] [PubMed] [Google Scholar]
  40. Pullen K. A., Rattray A. J., Champoux J. J. The sequence features important for plus strand priming by human immunodeficiency virus type 1 reverse transcriptase. J Biol Chem. 1993 Mar 25;268(9):6221–6227. [PubMed] [Google Scholar]
  41. Randolph C. A., Champoux J. J. The use of DNA and RNA oligonucleotides in hybrid structures with longer polynucleotide chains to probe the structural requirements for moloney murine leukemia virus plus strand priming. J Biol Chem. 1994 Jul 29;269(30):19207–19215. [PubMed] [Google Scholar]
  42. Ratmeyer L., Vinayak R., Zhong Y. Y., Zon G., Wilson W. D. Sequence specific thermodynamic and structural properties for DNA.RNA duplexes. Biochemistry. 1994 May 3;33(17):5298–5304. doi: 10.1021/bi00183a037. [DOI] [PubMed] [Google Scholar]
  43. Rattray A. J., Champoux J. J. Plus-strand priming by Moloney murine leukemia virus. The sequence features important for cleavage by RNase H. J Mol Biol. 1989 Aug 5;208(3):445–456. doi: 10.1016/0022-2836(89)90508-1. [DOI] [PubMed] [Google Scholar]
  44. Rattray A. J., Champoux J. J. The role of Moloney murine leukemia virus RNase H activity in the formation of plus-strand primers. J Virol. 1987 Sep;61(9):2843–2851. doi: 10.1128/jvi.61.9.2843-2851.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Risco C., Menéndez-Arias L., Copeland T. D., Pinto da Silva P., Oroszlan S. Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: nuclear import of nucleocapsid protein and integrase. J Cell Sci. 1995 Sep;108(Pt 9):3039–3050. doi: 10.1242/jcs.108.9.3039. [DOI] [PubMed] [Google Scholar]
  46. Schweizer M., Renne R., Neumann-Haefelin D. Structural analysis of proviral DNA in simian foamy virus (LK-3)-infected cells. Arch Virol. 1989;109(1-2):103–114. doi: 10.1007/BF01310521. [DOI] [PubMed] [Google Scholar]
  47. Shimotohno K., Temin H. M. Spontaneous variation and synthesis in the U3 region of the long terminal repeat of an avian retrovirus. J Virol. 1982 Jan;41(1):163–171. doi: 10.1128/jvi.41.1.163-171.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Takahashi H., Matsuda M., Kojima A., Sata T., Andoh T., Kurata T., Nagashima K., Hall W. W. Human immunodeficiency virus type 1 reverse transcriptase: enhancement of activity by interaction with cellular topoisomerase I. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5694–5698. doi: 10.1073/pnas.92.12.5694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Trono D. Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses. J Virol. 1992 Aug;66(8):4893–4900. doi: 10.1128/jvi.66.8.4893-4900.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Varela-Echavarría A., Garvey N., Preston B. D., Dougherty J. P. Comparison of Moloney murine leukemia virus mutation rate with the fidelity of its reverse transcriptase in vitro. J Biol Chem. 1992 Dec 5;267(34):24681–24688. [PubMed] [Google Scholar]
  51. Varmus H. E., Heasley S., Kung H. J., Oppermann H., Smith V. C., Bishop J. M., Shank P. R. Kinetics of synthesis, structure and purification of avian sarcoma virus-specific DNA made in the cytoplasm of acutely infected cells. J Mol Biol. 1978 Mar 25;120(1):55–82. doi: 10.1016/0022-2836(78)90295-4. [DOI] [PubMed] [Google Scholar]
  52. Williams K. J., Loeb L. A., Fry M. Synthesis of DNA by human immunodeficiency virus reverse transcriptase is preferentially blocked at template oligo(deoxyadenosine) tracts. J Biol Chem. 1990 Oct 25;265(30):18682–18689. [PubMed] [Google Scholar]
  53. Wu W., Henderson L. E., Copeland T. D., Gorelick R. J., Bosche W. J., Rein A., Levin J. G. Human immunodeficiency virus type 1 nucleocapsid protein reduces reverse transcriptase pausing at a secondary structure near the murine leukemia virus polypurine tract. J Virol. 1996 Oct;70(10):7132–7142. doi: 10.1128/jvi.70.10.7132-7142.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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