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. 1995 Jun;69(6):3938–3944. doi: 10.1128/jvi.69.6.3938-3944.1995

Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro.

M D Miller 1, B Wang 1, F D Bushman 1
PMCID: PMC189122  PMID: 7745750

Abstract

Despite intensive study, the mechanism by which many retroviruses complete reverse transcription has remained unclear. Most retroviruses and all lentiviruses fail to synthesize a full-length second strand of the viral cDNA (plus strand) efficiently in infected cells. For human immunodeficiency virus type 1, we find in synchronous infection experiments that full-length plus strands are rare (< 1% of products) at times when integration is likely taking place. Subviral nucleoprotein complexes containing such discontinuous cDNA can be extracted from infected cells and used to generate integration products in vitro. Analysis of such integration products using two-dimensional gel electrophoresis revealed that the discontinuous viral DNA was efficiently integrated into an added target DNA. These data support a model in which the discontinuities in the plus strand need not be sealed until after integration, potentially by the enzymes that are already thought to repair DNA gaps at the junctions between host and viral DNA.

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Selected References

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  1. 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]
  2. Blum H. E., Harris J. D., Ventura P., Walker D., Staskus K., Retzel E., Haase A. T. Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna. Virology. 1985 Apr 30;142(2):270–277. doi: 10.1016/0042-6822(85)90335-6. [DOI] [PubMed] [Google Scholar]
  3. Boone L. R., Skalka A. M. Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. I. Kinetics of synthesis and size of minus- and plus-strand transcripts. J Virol. 1981 Jan;37(1):109–116. doi: 10.1128/jvi.37.1.109-116.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boone L. R., Skalka A. M. Viral DNA synthesized in vitro by avian retrovirus particles permeabilized with melittin. II. Evidence for a strand displacement mechanism in plus-strand synthesis. J Virol. 1981 Jan;37(1):117–126. doi: 10.1128/jvi.37.1.117-126.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Correct integration of retroviral DNA in vitro. Cell. 1987 May 8;49(3):347–356. doi: 10.1016/0092-8674(87)90287-x. [DOI] [PubMed] [Google Scholar]
  6. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2525–2529. doi: 10.1073/pnas.86.8.2525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bukrinsky M. I., Haggerty S., Dempsey M. P., 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. doi: 10.1038/365666a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  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. Ellison V., Abrams H., Roe T., Lifson J., Brown P. Human immunodeficiency virus integration in a cell-free system. J Virol. 1990 Jun;64(6):2711–2715. doi: 10.1128/jvi.64.6.2711-2715.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Farnet C. M., Haseltine W. A. Circularization of human immunodeficiency virus type 1 DNA in vitro. J Virol. 1991 Dec;65(12):6942–6952. doi: 10.1128/jvi.65.12.6942-6952.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farnet C. M., Haseltine W. A. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol. 1991 Apr;65(4):1910–1915. doi: 10.1128/jvi.65.4.1910-1915.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Farnet C. M., Haseltine W. A. Integration of human immunodeficiency virus type 1 DNA in vitro. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4164–4168. doi: 10.1073/pnas.87.11.4164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fujiwara T., Mizuuchi K. Retroviral DNA integration: structure of an integration intermediate. Cell. 1988 Aug 12;54(4):497–504. doi: 10.1016/0092-8674(88)90071-2. [DOI] [PubMed] [Google Scholar]
  15. Gianni A. M., Weinberg R. A. Partially single-stranded form of free Moloney viral DNA. Nature. 1975 Jun 19;255(5510):646–648. doi: 10.1038/255646a0. [DOI] [PubMed] [Google Scholar]
  16. Gilboa E., Mitra S. W., Goff S., Baltimore D. A detailed model of reverse transcription and tests of crucial aspects. Cell. 1979 Sep;18(1):93–100. doi: 10.1016/0092-8674(79)90357-x. [DOI] [PubMed] [Google Scholar]
  17. Goff S. P. Retroviral reverse transcriptase: synthesis, structure, and function. J Acquir Immune Defic Syndr. 1990;3(8):817–831. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Kung H. J., Fung Y. K., Majors J. E., Bishop J. M., Varmus H. E. Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus. J Virol. 1981 Jan;37(1):127–138. doi: 10.1128/jvi.37.1.127-138.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Lee Y. M., Coffin J. M. Efficient autointegration of avian retrovirus DNA in vitro. J Virol. 1990 Dec;64(12):5958–5965. doi: 10.1128/jvi.64.12.5958-5965.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee Y. M., Coffin J. M. Relationship of avian retrovirus DNA synthesis to integration in vitro. Mol Cell Biol. 1991 Mar;11(3):1419–1430. doi: 10.1128/mcb.11.3.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pryciak P. M., Müller H. P., Varmus H. E. Simian virus 40 minichromosomes as targets for retroviral integration in vivo. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9237–9241. doi: 10.1073/pnas.89.19.9237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Whitcomb J. M., Hughes S. H. Retroviral reverse transcription and integration: progress and problems. Annu Rev Cell Biol. 1992;8:275–306. doi: 10.1146/annurev.cb.08.110192.001423. [DOI] [PubMed] [Google Scholar]

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