Abstract
Two different crystal structures of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalytic domain were analyzed for interactions at the enzyme active site. Gln-62 and Glu-92 interact with active-site residue Asp-64, and Lys-136 interacts with active-site residue Asp-116 across a dimer interface. Conservative and nonconservative substitutions were introduced at these positions to probe the roles of these interactions in HIV-1 integration. Purified mutant proteins were assayed for in vitro 3' processing, DNA strand transfer, and disintegration activities, and HIV-1 mutants were assayed for virion protein composition, reverse transcription, and infectivities in human cell lines. Each of the mutant IN proteins displayed wild-type disintegration activity, indicating that none of the interactions is essential for catalysis. Mutants carrying Gln or Ala for Glu-92 displayed wild-type activities, but substituting Lys for Glu-92 reduced in vitro 3' processing and DNA strand transfer activities 5- to 10-fold and yielded a replication-defective IN active-site mutant viral phenotype. Substituting Glu for Gln-62 reduced in vitro 3' processing and DNA strand transfer activities 5- to 10-fold without grossly affecting viral replication kinetics, suggesting that HIV-1 can replicate in T-cell lines with less than the wild-type level of IN activity. The relationship between IN solubility and HIV-1 replication was also investigated. We previously showed that substituting Lys for Phe-185 dramatically increased the solubility of recombinant IN but caused an HIV-1 particle assembly defect. Mutants carrying His at this position displayed increased solubility and wild-type replication kinetics, showing that increased IN solubility per se is not detrimental to virus growth.
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- 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]
- Ansari-Lari M. A., Donehower L. A., Gibbs R. A. Analysis of human immunodeficiency virus type 1 integrase mutants. Virology. 1995 Aug 1;211(1):332–335. doi: 10.1006/viro.1995.1412. [DOI] [PubMed] [Google Scholar]
- Bujacz G., Alexandratos J., Qing Z. L., Clément-Mella C., Wlodawer A. The catalytic domain of human immunodeficiency virus integrase: ordered active site in the F185H mutant. FEBS Lett. 1996 Dec 2;398(2-3):175–178. doi: 10.1016/s0014-5793(96)01236-7. [DOI] [PubMed] [Google Scholar]
- Bujacz G., Jaskólski M., Alexandratos J., Wlodawer A., Merkel G., Katz R. A., Skalka A. M. High-resolution structure of the catalytic domain of avian sarcoma virus integrase. J Mol Biol. 1995 Oct 20;253(2):333–346. doi: 10.1006/jmbi.1995.0556. [DOI] [PubMed] [Google Scholar]
- Bukovsky A., Göttlinger H. Lack of integrase can markedly affect human immunodeficiency virus type 1 particle production in the presence of an active viral protease. J Virol. 1996 Oct;70(10):6820–6825. doi: 10.1128/jvi.70.10.6820-6825.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Bushman F. D., Fujiwara T., Craigie R. Retroviral DNA integration directed by HIV integration protein in vitro. Science. 1990 Sep 28;249(4976):1555–1558. doi: 10.1126/science.2171144. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Dyda F., Hickman A. B., Jenkins T. M., Engelman A., Craigie R., Davies D. R. Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science. 1994 Dec 23;266(5193):1981–1986. doi: 10.1126/science.7801124. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Goodarzi G., Im G. J., Brackmann K., Grandgenett D. Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase. J Virol. 1995 Oct;69(10):6090–6097. doi: 10.1128/jvi.69.10.6090-6097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goulaouic H., Chow S. A. Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein. J Virol. 1996 Jan;70(1):37–46. doi: 10.1128/jvi.70.1.37-46.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grandgenett D. P., Goodarzi G. Folding of the multidomain human immunodeficiency virus type-I integrase. Protein Sci. 1994 Jun;3(6):888–897. doi: 10.1002/pro.5560030604. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jenkins T. M., Engelman A., Ghirlando R., Craigie R. A soluble active mutant of HIV-1 integrase: involvement of both the core and carboxyl-terminal domains in multimerization. J Biol Chem. 1996 Mar 29;271(13):7712–7718. doi: 10.1074/jbc.271.13.7712. [DOI] [PubMed] [Google Scholar]
- Jenkins T. M., Hickman A. B., Dyda F., Ghirlando R., Davies D. R., Craigie R. Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6057–6061. doi: 10.1073/pnas.92.13.6057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Leavitt A. D., Robles G., Alesandro N., Varmus H. E. Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J Virol. 1996 Feb;70(2):721–728. doi: 10.1128/jvi.70.2.721-728.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Masuda T., Planelles V., Krogstad P., Chen I. S. Genetic analysis of human immunodeficiency virus type 1 integrase and the U3 att site: unusual phenotype of mutants in the zinc finger-like domain. J Virol. 1995 Nov;69(11):6687–6696. doi: 10.1128/jvi.69.11.6687-6696.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rice P., Craigie R., Davies D. R. Retroviral integrases and their cousins. Curr Opin Struct Biol. 1996 Feb;6(1):76–83. doi: 10.1016/s0959-440x(96)80098-4. [DOI] [PubMed] [Google Scholar]
- Rice P., Mizuuchi K. Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration. Cell. 1995 Jul 28;82(2):209–220. doi: 10.1016/0092-8674(95)90308-9. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
- 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]
- Van Dyke M. W., Sirito M., Sawadogo M. Single-step purification of bacterially expressed polypeptides containing an oligo-histidine domain. Gene. 1992 Feb 1;111(1):99–104. doi: 10.1016/0378-1119(92)90608-r. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Weiss A., Wiskocil R. L., Stobo J. D. The role of T3 surface molecules in the activation of human T cells: a two-stimulus requirement for IL 2 production reflects events occurring at a pre-translational level. J Immunol. 1984 Jul;133(1):123–128. [PubMed] [Google Scholar]
- 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]
- Wiskerchen M., Muesing M. A. Identification and characterization of a temperature-sensitive mutant of human immunodeficiency virus type 1 by alanine scanning mutagenesis of the integrase gene. J Virol. 1995 Jan;69(1):597–601. doi: 10.1128/jvi.69.1.597-601.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]