Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication (original) (raw)

Abstract

The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site-directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes.

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Selected References

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  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker T. A., Luo L. Identification of residues in the Mu transposase essential for catalysis. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6654–6658. doi: 10.1073/pnas.91.14.6654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bushman F. D., Craigie R. Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1339–1343. doi: 10.1073/pnas.88.4.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bushman F. D., Engelman A., Palmer I., Wingfield P., Craigie R. Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3428–3432. doi: 10.1073/pnas.90.8.3428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cannon P. M., Wilson W., Byles E., Kingsman S. M., Kingsman A. J. Human immunodeficiency virus type 1 integrase: effect on viral replication of mutations at highly conserved residues. J Virol. 1994 Aug;68(8):4768–4775. doi: 10.1128/jvi.68.8.4768-4775.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chow S. A., Vincent K. A., Ellison V., Brown P. O. Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science. 1992 Feb 7;255(5045):723–726. doi: 10.1126/science.1738845. [DOI] [PubMed] [Google Scholar]
  7. Drelich M., Wilhelm R., Mous J. Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro. Virology. 1992 Jun;188(2):459–468. doi: 10.1016/0042-6822(92)90499-f. [DOI] [PubMed] [Google Scholar]
  8. Engelman A., Bushman F. D., Craigie R. Identification of discrete functional domains of HIV-1 integrase and their organization within an active multimeric complex. EMBO J. 1993 Aug;12(8):3269–3275. doi: 10.1002/j.1460-2075.1993.tb05996.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engelman A., Craigie R. Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro. J Virol. 1992 Nov;66(11):6361–6369. doi: 10.1128/jvi.66.11.6361-6369.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Engelman A., Hickman A. B., Craigie R. The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding. J Virol. 1994 Sep;68(9):5911–5917. doi: 10.1128/jvi.68.9.5911-5917.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engelman A., Mizuuchi K., Craigie R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell. 1991 Dec 20;67(6):1211–1221. doi: 10.1016/0092-8674(91)90297-c. [DOI] [PubMed] [Google Scholar]
  12. Fayet O., Ramond P., Polard P., Prère M. F., Chandler M. Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences? Mol Microbiol. 1990 Oct;4(10):1771–1777. doi: 10.1111/j.1365-2958.1990.tb00555.x. [DOI] [PubMed] [Google Scholar]
  13. Folks T., Benn S., Rabson A., Theodore T., Hoggan M. D., Martin M., Lightfoote M., Sell K. Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4539–4543. doi: 10.1073/pnas.82.13.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Freed E. O., Orenstein J. M., Buckler-White A. J., Martin M. A. Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J Virol. 1994 Aug;68(8):5311–5320. doi: 10.1128/jvi.68.8.5311-5320.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnson M. S., McClure M. A., Feng D. F., Gray J., Doolittle R. F. Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7648–7652. doi: 10.1073/pnas.83.20.7648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kalpana G. V., Goff S. P. Genetic analysis of homomeric interactions of human immunodeficiency virus type 1 integrase using the yeast two-hybrid system. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10593–10597. doi: 10.1073/pnas.90.22.10593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Katz R. A., Skalka A. M. The retroviral enzymes. Annu Rev Biochem. 1994;63:133–173. doi: 10.1146/annurev.bi.63.070194.001025. [DOI] [PubMed] [Google Scholar]
  19. Khan E., Mack J. P., Katz R. A., Kulkosky J., Skalka A. M. Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 1991 Feb 25;19(4):851–860. doi: 10.1093/nar/19.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Kulkosky J., Jones K. S., Katz R. A., Mack J. P., Skalka A. M. Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol. 1992 May;12(5):2331–2338. doi: 10.1128/mcb.12.5.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. LaFemina R. L., Schneider C. L., Robbins H. L., Callahan P. L., LeGrow K., Roth E., Schleif W. A., Emini E. A. Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells. J Virol. 1992 Dec;66(12):7414–7419. doi: 10.1128/jvi.66.12.7414-7419.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Le Grice S. F., Naas T., Wohlgensinger B., Schatz O. Subunit-selective mutagenesis indicates minimal polymerase activity in heterodimer-associated p51 HIV-1 reverse transcriptase. EMBO J. 1991 Dec;10(12):3905–3911. doi: 10.1002/j.1460-2075.1991.tb04960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Leavitt A. D., Shiue L., Varmus H. E. Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro. J Biol Chem. 1993 Jan 25;268(3):2113–2119. [PubMed] [Google Scholar]
  25. Lewis P., Hensel M., Emerman M. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J. 1992 Aug;11(8):3053–3058. doi: 10.1002/j.1460-2075.1992.tb05376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lightfoote M. M., Coligan J. E., Folks T. M., Fauci A. S., Martin M. A., Venkatesan S. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus. J Virol. 1986 Nov;60(2):771–775. doi: 10.1128/jvi.60.2.771-775.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lin T. H., Grandgenett D. P. Retrovirus integrase: identification of a potential leucine zipper motif. Protein Eng. 1991 Apr;4(4):435–441. doi: 10.1093/protein/4.4.435. [DOI] [PubMed] [Google Scholar]
  28. Lowe D. M., Parmar V., Kemp S. D., Larder B. A. Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. FEBS Lett. 1991 May 6;282(2):231–234. doi: 10.1016/0014-5793(91)80484-k. [DOI] [PubMed] [Google Scholar]
  29. Mazumder A., Engelman A., Craigie R., Fesen M., Pommier Y. Intermolecular disintegration and intramolecular strand transfer activities of wild-type and mutant HIV-1 integrase. Nucleic Acids Res. 1994 Mar 25;22(6):1037–1043. doi: 10.1093/nar/22.6.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Prince A. M., Horowitz B., Baker L., Shulman R. W., Ralph H., Valinsky J., Cundell A., Brotman B., Boehle W., Rey F. Failure of a human immunodeficiency virus (HIV) immune globulin to protect chimpanzees against experimental challenge with HIV. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6944–6948. doi: 10.1073/pnas.85.18.6944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Prince A. M., Reesink H., Pascual D., Horowitz B., Hewlett I., Murthy K. K., Cobb K. E., Eichberg J. W. Prevention of HIV infection by passive immunization with HIV immunoglobulin. AIDS Res Hum Retroviruses. 1991 Dec;7(12):971–973. doi: 10.1089/aid.1991.7.971. [DOI] [PubMed] [Google Scholar]
  32. Ross E. K., Buckler-White A. J., Rabson A. B., Englund G., Martin M. A. Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types. J Virol. 1991 Aug;65(8):4350–4358. doi: 10.1128/jvi.65.8.4350-4358.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roth M. J. Mutational analysis of the carboxyl terminus of the Moloney murine leukemia virus integration protein. J Virol. 1991 Apr;65(4):2141–2145. doi: 10.1128/jvi.65.4.2141-2145.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Roth M. J., Schwartzberg P., Tanese N., Goff S. P. Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting. J Virol. 1990 Oct;64(10):4709–4717. doi: 10.1128/jvi.64.10.4709-4717.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rowland S. J., Dyke K. G. Tn552, a novel transposable element from Staphylococcus aureus. Mol Microbiol. 1990 Jun;4(6):961–975. doi: 10.1111/j.1365-2958.1990.tb00669.x. [DOI] [PubMed] [Google Scholar]
  36. Rådström P., Sköld O., Swedberg G., Flensburg J., Roy P. H., Sundström L. Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J Bacteriol. 1994 Jun;176(11):3257–3268. doi: 10.1128/jb.176.11.3257-3268.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schauer M., Billich A. The N-terminal region of HIV-1 integrase is required for integration activity, but not for DNA-binding. Biochem Biophys Res Commun. 1992 Jun 30;185(3):874–880. doi: 10.1016/0006-291x(92)91708-x. [DOI] [PubMed] [Google Scholar]
  39. Sherman P. A., Fyfe J. A. Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5119–5123. doi: 10.1073/pnas.87.13.5119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shin C. G., Taddeo B., Haseltine W. A., Farnet C. M. Genetic analysis of the human immunodeficiency virus type 1 integrase protein. J Virol. 1994 Mar;68(3):1633–1642. doi: 10.1128/jvi.68.3.1633-1642.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Taddeo B., Haseltine W. A., Farnet C. M. Integrase mutants of human immunodeficiency virus type 1 with a specific defect in integration. J Virol. 1994 Dec;68(12):8401–8405. doi: 10.1128/jvi.68.12.8401-8405.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Telesnitsky A., Goff S. P. Two defective forms of reverse transcriptase can complement to restore retroviral infectivity. EMBO J. 1993 Nov;12(11):4433–4438. doi: 10.1002/j.1460-2075.1993.tb06128.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vincent K. A., Ellison V., Chow S. A., Brown P. O. Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations. J Virol. 1993 Jan;67(1):425–437. doi: 10.1128/jvi.67.1.425-437.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vink C., Oude Groeneger A. M., Plasterk R. H. Identification of the catalytic and DNA-binding region of the human immunodeficiency virus type I integrase protein. Nucleic Acids Res. 1993 Mar 25;21(6):1419–1425. doi: 10.1093/nar/21.6.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vink C., van Gent D. C., Elgersma Y., Plasterk R. H. Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage. J Virol. 1991 Sep;65(9):4636–4644. doi: 10.1128/jvi.65.9.4636-4644.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Willey R. L., Bonifacino J. S., Potts B. J., Martin M. A., Klausner R. D. Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9580–9584. doi: 10.1073/pnas.85.24.9580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Willey R. L., Smith D. H., Lasky L. A., Theodore T. S., Earl P. L., Moss B., Capon D. J., Martin M. A. In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity. J Virol. 1988 Jan;62(1):139–147. doi: 10.1128/jvi.62.1.139-147.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wiskerchen M., Muesing M. A. Human immunodeficiency virus type 1 integrase: effects of mutations on viral ability to integrate, direct viral gene expression from unintegrated viral DNA templates, and sustain viral propagation in primary cells. J Virol. 1995 Jan;69(1):376–386. doi: 10.1128/jvi.69.1.376-386.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Woerner A. M., Marcus-Sekura C. J. Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis. Nucleic Acids Res. 1993 Jul 25;21(15):3507–3511. doi: 10.1093/nar/21.15.3507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zack J. A., Arrigo S. J., Weitsman S. R., Go A. S., Haislip A., Chen I. S. HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell. 1990 Apr 20;61(2):213–222. doi: 10.1016/0092-8674(90)90802-l. [DOI] [PubMed] [Google Scholar]
  51. van Gent D. C., Groeneger A. A., Plasterk R. H. Mutational analysis of the integrase protein of human immunodeficiency virus type 2. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9598–9602. doi: 10.1073/pnas.89.20.9598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. van Gent D. C., Vink C., Groeneger A. A., Plasterk R. H. Complementation between HIV integrase proteins mutated in different domains. EMBO J. 1993 Aug;12(8):3261–3267. doi: 10.1002/j.1460-2075.1993.tb05995.x. [DOI] [PMC free article] [PubMed] [Google Scholar]