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Journal of Virology logoLink to Journal of Virology
. 1995 Nov;69(11):6687–6696. doi: 10.1128/jvi.69.11.6687-6696.1995

Genetic analysis of human immunodeficiency virus type 1 integrase and the U3 att site: unusual phenotype of mutants in the zinc finger-like domain.

T Masuda 1, V Planelles 1, P Krogstad 1, I S Chen 1
PMCID: PMC189578  PMID: 7474078

Abstract

Retroviral integration is the step which leads to establishment of the provirus, cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild-type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression. Interestingly, we found that three different HIV-1 constructs bearing point mutations in the zinc finger-like domain synthesized much lower levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of reverse transcriptase activity. These data indicate that in addition to integration, IN may have an effect on the proper course of events in the viral life cycle that precede integration.

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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. 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]
  3. 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]
  4. Burke C. J., Sanyal G., Bruner M. W., Ryan J. A., LaFemina R. L., Robbins H. L., Zeft A. S., Middaugh C. R., Cordingley M. G. Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase. J Biol Chem. 1992 May 15;267(14):9639–9644. [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Bushman F. D., Wang B. Rous sarcoma virus integrase protein: mapping functions for catalysis and substrate binding. J Virol. 1994 Apr;68(4):2215–2223. doi: 10.1128/jvi.68.4.2215-2223.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Camerini D., Planelles V., Chen I. S. CD26 antigen and HIV fusion? Science. 1994 May 20;264(5162):1160–1165. doi: 10.1126/science.7909961. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Chen B. K., Saksela K., Andino R., Baltimore D. Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferase-encoding viruses. J Virol. 1994 Feb;68(2):654–660. doi: 10.1128/jvi.68.2.654-660.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chow S. A., Brown P. O. Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates. J Virol. 1994 Jun;68(6):3896–3907. doi: 10.1128/jvi.68.6.3896-3907.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Colicelli J., Goff S. P. Mutants and pseudorevertants of Moloney murine leukemia virus with alterations at the integration site. Cell. 1985 Sep;42(2):573–580. doi: 10.1016/0092-8674(85)90114-x. [DOI] [PubMed] [Google Scholar]
  14. Colicelli J., Goff S. P. Sequence and spacing requirements of a retrovirus integration site. J Mol Biol. 1988 Jan 5;199(1):47–59. doi: 10.1016/0022-2836(88)90378-6. [DOI] [PubMed] [Google Scholar]
  15. Donehower L. A. Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3' region of pol. J Virol. 1988 Nov;62(11):3958–3964. doi: 10.1128/jvi.62.11.3958-3964.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Donehower L. A., Varmus H. E. A mutant murine leukemia virus with a single missense codon in pol is defective in a function affecting integration. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6461–6465. doi: 10.1073/pnas.81.20.6461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dougherty J. P., Wisniewski R., Yang S. L., Rhode B. W., Temin H. M. New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors. J Virol. 1989 Jul;63(7):3209–3212. doi: 10.1128/jvi.63.7.3209-3212.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Engelman A., Englund G., Orenstein J. M., Martin M. A., Craigie R. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol. 1995 May;69(5):2729–2736. doi: 10.1128/jvi.69.5.2729-2736.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Englund G., Theodore T. S., Freed E. O., Engelman A., Martin M. A. Integration is required for productive infection of monocyte-derived macrophages by human immunodeficiency virus type 1. J Virol. 1995 May;69(5):3216–3219. doi: 10.1128/jvi.69.5.3216-3219.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goff S. P. Genetics of retroviral integration. Annu Rev Genet. 1992;26:527–544. doi: 10.1146/annurev.ge.26.120192.002523. [DOI] [PubMed] [Google Scholar]
  25. Harris J. D., Blum H., Scott J., Traynor B., Ventura P., Haase A. Slow virus visna: reproduction in vitro of virus from extrachromosomal DNA. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7212–7215. doi: 10.1073/pnas.81.22.7212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hippenmeyer P. J., Grandgenett D. P. Requirement of the avian retrovirus pp32 DNA binding protein domain for replication. Virology. 1984 Sep;137(2):358–370. doi: 10.1016/0042-6822(84)90228-9. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Jonsson C. B., Roth M. J. Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration. J Virol. 1993 Sep;67(9):5562–5571. doi: 10.1128/jvi.67.9.5562-5571.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jowett J. B., Planelles V., Poon B., Shah N. P., Chen M. L., Chen I. S. The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2 + M phase of the cell cycle. J Virol. 1995 Oct;69(10):6304–6313. doi: 10.1128/jvi.69.10.6304-6313.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kaplan A. H., Krogstad P., Kempf D. J., Norbeck D. W., Swanstrom R. Human immunodeficiency virus type 1 virions composed of unprocessed Gag and Gag-Pol precursors are capable of reverse transcribing viral genomic RNA. Antimicrob Agents Chemother. 1994 Dec;38(12):2929–2933. doi: 10.1128/aac.38.12.2929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Katz R. A., Merkel G., Kulkosky J., Leis J., Skalka A. M. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell. 1990 Oct 5;63(1):87–95. doi: 10.1016/0092-8674(90)90290-u. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Koyanagi Y., Miles S., Mitsuyasu R. T., Merrill J. E., Vinters H. V., Chen I. S. Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science. 1987 May 15;236(4803):819–822. doi: 10.1126/science.3646751. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Kulkosky J., Skalka A. M. HIV DNA integration: observations and interferences. J Acquir Immune Defic Syndr. 1990;3(9):839–851. [PubMed] [Google Scholar]
  37. LaFemina R. L., Callahan P. L., Cordingley M. G. Substrate specificity of recombinant human immunodeficiency virus integrase protein. J Virol. 1991 Oct;65(10):5624–5630. doi: 10.1128/jvi.65.10.5624-5630.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Lawn R. M., Efstratiadis A., O'Connell C., Maniatis T. The nucleotide sequence of the human beta-globin gene. Cell. 1980 Oct;21(3):647–651. doi: 10.1016/0092-8674(80)90428-6. [DOI] [PubMed] [Google Scholar]
  40. Leavitt A. D., Rose R. B., Varmus H. E. Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae. J Virol. 1992 Apr;66(4):2359–2368. doi: 10.1128/jvi.66.4.2359-2368.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. McEuen A. R., Edwards B., Koepke K. A., Ball A. E., Jennings B. A., Wolstenholme A. J., Danson M. J., Hough D. W. Zinc binding by retroviral integrase. Biochem Biophys Res Commun. 1992 Dec 15;189(2):813–818. doi: 10.1016/0006-291x(92)92275-3. [DOI] [PubMed] [Google Scholar]
  43. Murphy J. E., Goff S. P. A mutation at one end of Moloney murine leukemia virus DNA blocks cleavage of both ends by the viral integrase in vivo. J Virol. 1992 Aug;66(8):5092–5095. doi: 10.1128/jvi.66.8.5092-5095.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Murphy J. E., Goff S. P. Construction and analysis of deletion mutations in the U5 region of Moloney murine leukemia virus: effects on RNA packaging and reverse transcription. J Virol. 1989 Jan;63(1):319–327. doi: 10.1128/jvi.63.1.319-327.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Panganiban A. T., Temin H. M. The terminal nucleotides of retrovirus DNA are required for integration but not virus production. Nature. 1983 Nov 10;306(5939):155–160. doi: 10.1038/306155a0. [DOI] [PubMed] [Google Scholar]
  46. Planelles V., Bachelerie F., Jowett J. B., Haislip A., Xie Y., Banooni P., Masuda T., Chen I. S. Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr. J Virol. 1995 Sep;69(9):5883–5889. doi: 10.1128/jvi.69.9.5883-5889.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Planelles V., Haislip A., Withers-Ward E. S., Stewart S. A., Xie Y., Shah N. P., Chen I. S. A new reporter system for detection of retroviral infection. Gene Ther. 1995 Aug;2(6):369–376. [PubMed] [Google Scholar]
  48. Rogel M. E., Wu L. I., Emerman M. The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection. J Virol. 1995 Feb;69(2):882–888. doi: 10.1128/jvi.69.2.882-888.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Roth M. J., Schwartzberg P. L., Goff S. P. Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence. Cell. 1989 Jul 14;58(1):47–54. doi: 10.1016/0092-8674(89)90401-7. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Sakai H., Kawamura M., Sakuragi J., Sakuragi S., Shibata R., Ishimoto A., Ono N., Ueda S., Adachi A. Integration is essential for efficient gene expression of human immunodeficiency virus type 1. J Virol. 1993 Mar;67(3):1169–1174. doi: 10.1128/jvi.67.3.1169-1174.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Schwartzberg P., Colicelli J., Goff S. P. Construction and analysis of deletion mutations in the pol gene of Moloney murine leukemia virus: a new viral function required for productive infection. Cell. 1984 Jul;37(3):1043–1052. doi: 10.1016/0092-8674(84)90439-2. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. 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]
  56. Stevenson M., Haggerty S., Lamonica C. A., Meier C. M., Welch S. K., Wasiak A. J. Integration is not necessary for expression of human immunodeficiency virus type 1 protein products. J Virol. 1990 May;64(5):2421–2425. doi: 10.1128/jvi.64.5.2421-2425.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. 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]
  59. 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]
  60. 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]
  61. 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]
  62. Zack J. A., Haislip A. M., Krogstad P., Chen I. S. Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle. J Virol. 1992 Mar;66(3):1717–1725. doi: 10.1128/jvi.66.3.1717-1725.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. 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]
  65. 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]

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