Abstract
Certain amino acid substitutions in the reverse transcriptase (RT), including D67N, K70R, T215Y, and K219Q, cause high-level resistance of human immunodeficiency virus type 1 (HIV-1) to zidovudine (3'-azidothymidine; AZT) and appear to approximate the template strand of the enzyme-template-primer complex in structural models. We studied whether this set of mutations altered RT-template-primer interaction as well as their effect on virus replication in the absence of inhibitor. When in vitro polymerization was limited to a single association of an RT with an oligodeoxynucleotide-primed heteropolymeric RNA template (a single processive cycle), recombinant-expressed mutant 67/70/215/219 RT synthesized 5- to 10-fold more high-molecular-weight DNA products (>200 nucleotides in length) than wild-type RT. This advantage was maintained as deoxynucleoside triphosphate (dNTP) concentrations were decreased to limiting levels. In contrast, no difference was seen between wild-type and mutant RTs under conditions allowing repeated associations of enzyme with template-primer. Because intracellular dNTP concentrations are low prior to mitogenic stimulation, we compared replication of mutant 67/70/215/219 virus and wild-type virus in peripheral blood mononuclear cells (PBMC) stimulated before and after infection. In the absence of inhibitor, mutant 67/70/215/219 virus had a replication advantage in PBMC stimulated with phytohemagglutinin and interleukin-2 after infection, but virus replication was similar in PBMC stimulated before infection in vitro. The results confirm that RT mutations D67N, K70R, T215Y, and K219Q affect an enzyme-template-primer interaction in vitro and suggest that such substitutions may affect HIV-1 pathogenesis during therapy by increasing viral replication capacity in cells stimulated after infection.
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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]
- Boucher C. A., van Leeuwen R., Kellam P., Schipper P., Tijnagel J., Lange J. M., Larder B. A. Effects of discontinuation of zidovudine treatment on zidovudine sensitivity of human immunodeficiency virus type 1 isolates. Antimicrob Agents Chemother. 1993 Jul;37(7):1525–1530. doi: 10.1128/aac.37.7.1525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boyer P. L., Tantillo C., Jacobo-Molina A., Nanni R. G., Ding J., Arnold E., Hughes S. H. Sensitivity of wild-type human immunodeficiency virus type 1 reverse transcriptase to dideoxynucleotides depends on template length; the sensitivity of drug-resistant mutants does not. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4882–4886. doi: 10.1073/pnas.91.11.4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bukrinsky M. I., Stanwick T. L., Dempsey M. P., Stevenson M. Quiescent T lymphocytes as an inducible virus reservoir in HIV-1 infection. Science. 1991 Oct 18;254(5030):423–427. doi: 10.1126/science.1925601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chow Y. K., Hirsch M. S., Merrill D. P., Bechtel L. J., Eron J. J., Kaplan J. C., D'Aquila R. T. Use of evolutionary limitations of HIV-1 multidrug resistance to optimize therapy. Nature. 1993 Feb 18;361(6413):650–654. doi: 10.1038/361650a0. [DOI] [PubMed] [Google Scholar]
- Coffin J. M. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science. 1995 Jan 27;267(5197):483–489. doi: 10.1126/science.7824947. [DOI] [PubMed] [Google Scholar]
- D'Aquila R. T., Johnson V. A., Welles S. L., Japour A. J., Kuritzkes D. R., DeGruttola V., Reichelderfer P. S., Coombs R. W., Crumpacker C. S., Kahn J. O. Zidovudine resistance and HIV-1 disease progression during antiretroviral therapy. AIDS Clinical Trials Group Protocol 116B/117 Team and the Virology Committee Resistance Working Group. Ann Intern Med. 1995 Mar 15;122(6):401–408. doi: 10.7326/0003-4819-122-6-199503150-00001. [DOI] [PubMed] [Google Scholar]
- D'Aquila R. T., Summers W. C. HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction. J Acquir Immune Defic Syndr. 1989;2(6):579–587. [PubMed] [Google Scholar]
- Eron J. J., Chow Y. K., Caliendo A. M., Videler J., Devore K. M., Cooley T. P., Liebman H. A., Kaplan J. C., Hirsch M. S., D'Aquila R. T. pol mutations conferring zidovudine and didanosine resistance with different effects in vitro yield multiply resistant human immunodeficiency virus type 1 isolates in vivo. Antimicrob Agents Chemother. 1993 Jul;37(7):1480–1487. doi: 10.1128/aac.37.7.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eron J. J., Gorczyca P., Kaplan J. C., D'Aquila R. T. Susceptibility testing by polymerase chain reaction DNA quantitation: a method to measure drug resistance of human immunodeficiency virus type 1 isolates. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3241–3245. doi: 10.1073/pnas.89.8.3241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gao W. Y., Agbaria R., Driscoll J. S., Mitsuya H. Divergent anti-human immunodeficiency virus activity and anabolic phosphorylation of 2',3'-dideoxynucleoside analogs in resting and activated human cells. J Biol Chem. 1994 Apr 29;269(17):12633–12638. [PubMed] [Google Scholar]
- Gao W. Y., Cara A., Gallo R. C., Lori F. Low levels of deoxynucleotides in peripheral blood lymphocytes: a strategy to inhibit human immunodeficiency virus type 1 replication. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8925–8928. doi: 10.1073/pnas.90.19.8925. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gibbs J. S., Regier D. A., Desrosiers R. C. Construction and in vitro properties of HIV-1 mutants with deletions in "nonessential" genes. AIDS Res Hum Retroviruses. 1994 Apr;10(4):343–350. doi: 10.1089/aid.1994.10.343. [DOI] [PubMed] [Google Scholar]
- Hammer S., Crumpacker C., D'Aquila R., Jackson B., Lathey J., Livnat D., Reichelderfer P. Use of virologic assays for detection of human immunodeficiency virus in clinical trials: recommendations of the AIDS Clinical Trials Group Virology Committee. J Clin Microbiol. 1993 Oct;31(10):2557–2564. doi: 10.1128/jcm.31.10.2557-2564.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ho D. D., Neumann A. U., Perelson A. S., Chen W., Leonard J. M., Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature. 1995 Jan 12;373(6510):123–126. doi: 10.1038/373123a0. [DOI] [PubMed] [Google Scholar]
- Holodniy M., Mole L., Margolis D., Moss J., Dong H., Boyer E., Urdea M., Kolberg J., Eastman S. Determination of human immunodeficiency virus RNA in plasma and cellular viral DNA genotypic zidovudine resistance and viral load during zidovudine-didanosine combination therapy. J Virol. 1995 Jun;69(6):3510–3516. doi: 10.1128/jvi.69.6.3510-3516.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huber H. E., McCoy J. M., Seehra J. S., Richardson C. C. Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. J Biol Chem. 1989 Mar 15;264(8):4669–4678. [PubMed] [Google Scholar]
- Jackson J. B., Coombs R. W., Sannerud K., Rhame F. S., Balfour H. H., Jr Rapid and sensitive viral culture method for human immunodeficiency virus type 1. J Clin Microbiol. 1988 Jul;26(7):1416–1418. doi: 10.1128/jcm.26.7.1416-1418.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacobo-Molina A., Ding J., Nanni R. G., Clark A. D., Jr, Lu X., Tantillo C., Williams R. L., Kamer G., Ferris A. L., Clark P. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6320–6324. doi: 10.1073/pnas.90.13.6320. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Japour A. J., Mayers D. L., Johnson V. A., Kuritzkes D. R., Beckett L. A., Arduino J. M., Lane J., Black R. J., Reichelderfer P. S., D'Aquila R. T. Standardized peripheral blood mononuclear cell culture assay for determination of drug susceptibilities of clinical human immunodeficiency virus type 1 isolates. The RV-43 Study Group, the AIDS Clinical Trials Group Virology Committee Resistance Working Group. Antimicrob Agents Chemother. 1993 May;37(5):1095–1101. doi: 10.1128/aac.37.5.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Japour A. J., Welles S., D'Aquila R. T., Johnson V. A., Richman D. D., Coombs R. W., Reichelderfer P. S., Kahn J. O., Crumpacker C. S., Kuritzkes D. R. Prevalence and clinical significance of zidovudine resistance mutations in human immunodeficiency virus isolated from patients after long-term zidovudine treatment. AIDS Clinical Trials Group 116B/117 Study Team and the Virology Committee Resistance Working Group. J Infect Dis. 1995 May;171(5):1172–1179. doi: 10.1093/infdis/171.5.1172. [DOI] [PubMed] [Google Scholar]
- Johnson V. A., Merrill D. P., Videler J. A., Chou T. C., Byington R. E., Eron J. J., D'Aquila R. T., Hirsch M. S. Two-drug combinations of zidovudine, didanosine, and recombinant interferon-alpha A inhibit replication of zidovudine-resistant human immunodeficiency virus type 1 synergistically in vitro. J Infect Dis. 1991 Oct;164(4):646–655. doi: 10.1093/infdis/164.4.646. [DOI] [PubMed] [Google Scholar]
- Kohlstaedt L. A., Steitz T. A. Reverse transcriptase of human immunodeficiency virus can use either human tRNA(3Lys) or Escherichia coli tRNA(2Gln) as a primer in an in vitro primer-utilization assay. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9652–9656. doi: 10.1073/pnas.89.20.9652. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kohlstaedt L. A., Wang J., Friedman J. M., Rice P. A., Steitz T. A. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 1992 Jun 26;256(5065):1783–1790. doi: 10.1126/science.1377403. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Lacey S. F., Reardon J. E., Furfine E. S., Kunkel T. A., Bebenek K., Eckert K. A., Kemp S. D., Larder B. A. Biochemical studies on the reverse transcriptase and RNase H activities from human immunodeficiency virus strains resistant to 3'-azido-3'-deoxythymidine. J Biol Chem. 1992 Aug 5;267(22):15789–15794. [PubMed] [Google Scholar]
- Larder B. A., Darby G., Richman D. D. HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy. Science. 1989 Mar 31;243(4899):1731–1734. doi: 10.1126/science.2467383. [DOI] [PubMed] [Google Scholar]
- Larder B. A., Kemp S. D., Harrigan P. R. Potential mechanism for sustained antiretroviral efficacy of AZT-3TC combination therapy. Science. 1995 Aug 4;269(5224):696–699. doi: 10.1126/science.7542804. [DOI] [PubMed] [Google Scholar]
- Larder B. A., Kemp S. D. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science. 1989 Dec 1;246(4934):1155–1158. doi: 10.1126/science.2479983. [DOI] [PubMed] [Google Scholar]
- Loveday C., Kaye S., Tenant-Flowers M., Semple M., Ayliffe U., Weller I. V., Tedder R. S. HIV-1 RNA serum-load and resistant viral genotypes during early zidovudine therapy. Lancet. 1995 Apr 1;345(8953):820–824. doi: 10.1016/s0140-6736(95)92963-0. [DOI] [PubMed] [Google Scholar]
- Majumdar C., Abbotts J., Broder S., Wilson S. H. Studies on the mechanism of human immunodeficiency virus reverse transcriptase. Steady-state kinetics, processivity, and polynucleotide inhibition. J Biol Chem. 1988 Oct 25;263(30):15657–15665. [PubMed] [Google Scholar]
- Martin J. L., Wilson J. E., Haynes R. L., Furman P. A. Mechanism of resistance of human immunodeficiency virus type 1 to 2',3'-dideoxyinosine. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6135–6139. doi: 10.1073/pnas.90.13.6135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McDougal J. S., Mawle A., Cort S. P., Nicholson J. K., Cross G. D., Scheppler-Campbell J. A., Hicks D., Sligh J. Cellular tropism of the human retrovirus HTLV-III/LAV. I. Role of T cell activation and expression of the T4 antigen. J Immunol. 1985 Nov;135(5):3151–3162. [PubMed] [Google Scholar]
- Nájera I., Holguín A., Quiñones-Mateu M. E., Muñoz-Fernández M. A., Nájera R., López-Galíndez C., Domingo E. Pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy. J Virol. 1995 Jan;69(1):23–31. doi: 10.1128/jvi.69.1.23-31.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nájera I., Richman D. D., Olivares I., Rojas J. M., Peinado M. A., Perucho M., Nájera R., López-Galíndez C. Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates. AIDS Res Hum Retroviruses. 1994 Nov;10(11):1479–1488. doi: 10.1089/aid.1994.10.1479. [DOI] [PubMed] [Google Scholar]
- Patterson B. K., Till M., Otto P., Goolsby C., Furtado M. R., McBride L. J., Wolinsky S. M. Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science. 1993 May 14;260(5110):976–979. doi: 10.1126/science.8493534. [DOI] [PubMed] [Google Scholar]
- Rashtchian A., Buchman G. W., Schuster D. M., Berninger M. S. Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning. Anal Biochem. 1992 Oct;206(1):91–97. doi: 10.1016/s0003-2697(05)80015-6. [DOI] [PubMed] [Google Scholar]
- Rashtchian A., Thornton C. G., Heidecker G. A novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase. PCR Methods Appl. 1992 Nov;2(2):124–130. doi: 10.1101/gr.2.2.124. [DOI] [PubMed] [Google Scholar]
- Ratner L., Fisher A., Jagodzinski L. L., Mitsuya H., Liou R. S., Gallo R. C., Wong-Staal F. Complete nucleotide sequences of functional clones of the AIDS virus. AIDS Res Hum Retroviruses. 1987 Spring;3(1):57–69. doi: 10.1089/aid.1987.3.57. [DOI] [PubMed] [Google Scholar]
- Reardon J. E., Miller W. H. Human immunodeficiency virus reverse transcriptase. Substrate and inhibitor kinetics with thymidine 5'-triphosphate and 3'-azido-3'-deoxythymidine 5'-triphosphate. J Biol Chem. 1990 Nov 25;265(33):20302–20307. [PubMed] [Google Scholar]
- Reddy M. K., Weitzel S. E., von Hippel P. H. Processive proofreading is intrinsic to T4 DNA polymerase. J Biol Chem. 1992 Jul 15;267(20):14157–14166. [PubMed] [Google Scholar]
- Shirasaka T., Chokekijchai S., Yamada A., Gosselin G., Imbach J. L., Mitsuya H. Comparative analysis of anti-human immunodeficiency virus type 1 activities of dideoxynucleoside analogs in resting and activated peripheral blood mononuclear cells. Antimicrob Agents Chemother. 1995 Nov;39(11):2555–2559. doi: 10.1128/aac.39.11.2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spina C. A., Guatelli J. C., Richman D. D. Establishment of a stable, inducible form of human immunodeficiency virus type 1 DNA in quiescent CD4 lymphocytes in vitro. J Virol. 1995 May;69(5):2977–2988. doi: 10.1128/jvi.69.5.2977-2988.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- St Clair M. H., Martin J. L., Tudor-Williams G., Bach M. C., Vavro C. L., King D. M., Kellam P., Kemp S. D., Larder B. A. Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase. Science. 1991 Sep 27;253(5027):1557–1559. doi: 10.1126/science.1716788. [DOI] [PubMed] [Google Scholar]
- Stevenson M., Stanwick T. L., Dempsey M. P., Lamonica C. A. HIV-1 replication is controlled at the level of T cell activation and proviral integration. EMBO J. 1990 May;9(5):1551–1560. doi: 10.1002/j.1460-2075.1990.tb08274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tang S., Patterson B., Levy J. A. Highly purified quiescent human peripheral blood CD4+ T cells are infectible by human immunodeficiency virus but do not release virus after activation. J Virol. 1995 Sep;69(9):5659–5665. doi: 10.1128/jvi.69.9.5659-5665.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tantillo C., Ding J., Jacobo-Molina A., Nanni R. G., Boyer P. L., Hughes S. H., Pauwels R., Andries K., Janssen P. A., Arnold E. Locations of anti-AIDS drug binding sites and resistance mutations in the three-dimensional structure of HIV-1 reverse transcriptase. Implications for mechanisms of drug inhibition and resistance. J Mol Biol. 1994 Oct 28;243(3):369–387. doi: 10.1006/jmbi.1994.1665. [DOI] [PubMed] [Google Scholar]
- Wei X., Ghosh S. K., Taylor M. E., Johnson V. A., Emini E. A., Deutsch P., Lifson J. D., Bonhoeffer S., Nowak M. A., Hahn B. H. Viral dynamics in human immunodeficiency virus type 1 infection. Nature. 1995 Jan 12;373(6510):117–122. doi: 10.1038/373117a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Zhang D., Caliendo A. M., Eron J. J., DeVore K. M., Kaplan J. C., Hirsch M. S., D'Aquila R. T. Resistance to 2',3'-dideoxycytidine conferred by a mutation in codon 65 of the human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother. 1994 Feb;38(2):282–287. doi: 10.1128/aac.38.2.282. [DOI] [PMC free article] [PubMed] [Google Scholar]
- von Schwedler U., Kornbluth R. S., Trono D. The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6992–6996. doi: 10.1073/pnas.91.15.6992. [DOI] [PMC free article] [PubMed] [Google Scholar]