Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1996 Nov;40(11):2535–2541. doi: 10.1128/aac.40.11.2535

Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening procedure for their detection.

M B Vasudevachari 1, Y M Zhang 1, H Imamichi 1, T Imamichi 1, J Falloon 1, N P Salzman 1
PMCID: PMC163570  PMID: 8913459

Abstract

Patient human immunodeficiency virus type 1 (HIV-1) isolates that are resistant to protease inhibitors may contain amino acid substitutions L10I/V, M46L/I, G-48V, L63P, V82A/F/T, I84V, and L90M in the protease gene. Substitutions at positions 82 and/or 90 occur in variants that display high levels of resistance to certain protease inhibitors. Nucleotide substitutions at these two sites also lead to the loss of two HindII restriction enzyme digestion sites, and these changes make possible a rapid procedure for the detection of drug-resistant variants in patients on protease inhibitor therapy. This procedure was used to detect the emergence of mutated viruses at various times after the initiation of therapy with the HIV-1 protease inhibitor indinavir. The method includes viral RNA isolation from plasma and reverse transcription PCR amplification of the protease gene with fluorescence-tagged primers. The PCR product is digested with HindII, the cleavage products are separated on a urea-acrylamide gel in a DNA sequencer, and the extent of cleavage is automatically analyzed with commercially available software. In viruses from 34 blood samples from four patients, mutations leading to an amino acid change at residue 82 appeared as early as 6 weeks after the start of therapy and persisted throughout the course of the study period (48 weeks). Mutations leading to double substitutions at residues 82 and 90 were seen at a lower frequency and appeared later than the change at position 82. The changes detected by restriction enzyme cleavage were confirmed by DNA sequencing of the cloned protease genes by reverse transcription PCR amplification of viral RNA from isolates in plasma. In addition to the changes at positions 82 and 90, we have identified M46L/I, G48V, and I54V substitutions in isolates derived from indinavir-treated patients. HindII analysis of uncloned, PCR-amplified DNA offers a rapid screening procedure for the detection of virus isolates containing mutations at amino acid residues 82 and 90 in the HIV-1 protease gene. By using other restriction enzymes, the same method can be used to detect additional protease drug-resistant variants and is generally applicable for the detection of mutations.

Full Text

The Full Text of this article is available as a PDF (253.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baldwin E. T., Bhat T. N., Liu B., Pattabiraman N., Erickson J. W. Structural basis of drug resistance for the V82A mutant of HIV-1 proteinase. Nat Struct Biol. 1995 Mar;2(3):244–249. doi: 10.1038/nsb0395-244. [DOI] [PubMed] [Google Scholar]
  2. Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen Z., Li Y., Schock H. B., Hall D., Chen E., Kuo L. C. Three-dimensional structure of a mutant HIV-1 protease displaying cross-resistance to all protease inhibitors in clinical trials. J Biol Chem. 1995 Sep 15;270(37):21433–21436. doi: 10.1074/jbc.270.37.21433. [DOI] [PubMed] [Google Scholar]
  4. Collier A. C., Coombs R. W., Schoenfeld D. A., Bassett R. L., Timpone J., Baruch A., Jones M., Facey K., Whitacre C., McAuliffe V. J. Treatment of human immunodeficiency virus infection with saquinavir, zidovudine, and zalcitabine. AIDS Clinical Trials Group. N Engl J Med. 1996 Apr 18;334(16):1011–1017. doi: 10.1056/NEJM199604183341602. [DOI] [PubMed] [Google Scholar]
  5. Condra J. H., Schleif W. A., Blahy O. M., Gabryelski L. J., Graham D. J., Quintero J. C., Rhodes A., Robbins H. L., Roth E., Shivaprakash M. In vivo emergence of HIV-1 variants resistant to multiple protease inhibitors. Nature. 1995 Apr 6;374(6522):569–571. doi: 10.1038/374569a0. [DOI] [PubMed] [Google Scholar]
  6. Eastman P. S., Boyer E., Mole L., Kolberg J., Urdea M., Holodniy M. Nonisotopic hybridization assay for determination of relative amounts of genotypic human immunodeficiency virus type 1 zidovudine resistance. J Clin Microbiol. 1995 Oct;33(10):2777–2780. doi: 10.1128/jcm.33.10.2777-2780.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eastman P. S., Urdea M., Besemer D., Stempien M., Kolberg J. Comparison of selective polymerase chain reaction primers and differential probe hybridization of polymerase chain reaction products for determination of relative amounts of codon 215 mutant and wild-type HIV-1 populations. J Acquir Immune Defic Syndr Hum Retrovirol. 1995 Jul 1;9(3):264–273. [PubMed] [Google Scholar]
  8. Eberle J., Bechowsky B., Rose D., Hauser U., von der Helm K., Gürtler L., Nitschko H. Resistance of HIV type 1 to proteinase inhibitor Ro 31-8959. AIDS Res Hum Retroviruses. 1995 Jun;11(6):671–676. doi: 10.1089/aid.1995.11.671. [DOI] [PubMed] [Google Scholar]
  9. Fontenot G., Johnston K., Cohen J. C., Gallaher W. R., Robinson J., Luftig R. B. PCR amplification of HIV-1 proteinase sequences directly from lab isolates allows determination of five conserved domains. Virology. 1992 Sep;190(1):1–10. doi: 10.1016/0042-6822(92)91186-x. [DOI] [PubMed] [Google Scholar]
  10. Gulnik S. V., Suvorov L. I., Liu B., Yu B., Anderson B., Mitsuya H., Erickson J. W. Kinetic characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug pressure. Biochemistry. 1995 Jul 25;34(29):9282–9287. doi: 10.1021/bi00029a002. [DOI] [PubMed] [Google Scholar]
  11. Ho D. D., Toyoshima T., Mo H., Kempf D. J., Norbeck D., Chen C. M., Wideburg N. E., Burt S. K., Erickson J. W., Singh M. K. Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor. J Virol. 1994 Mar;68(3):2016–2020. doi: 10.1128/jvi.68.3.2016-2020.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobsen H., Yasargil K., Winslow D. L., Craig J. C., Kröhn A., Duncan I. B., Mous J. Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro 31-8959. Virology. 1995 Jan 10;206(1):527–534. doi: 10.1016/s0042-6822(95)80069-7. [DOI] [PubMed] [Google Scholar]
  13. Jung M., Agut H., Candotti D., Ingrand D., Katlama C., Huraux J. M. Susceptibility of HIV-1 isolates to zidovudine: correlation between widely applicable culture test and PCR analysis. J Acquir Immune Defic Syndr. 1992;5(4):359–364. [PubMed] [Google Scholar]
  14. Kaplan A. H., Michael S. F., Wehbie R. S., Knigge M. F., Paul D. A., Everitt L., Kempf D. J., Norbeck D. W., Erickson J. W., Swanstrom R. Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5597–5601. doi: 10.1073/pnas.91.12.5597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. King R. W., Winslow D. L., Garber S., Scarnati H. T., Bachelor L., Stack S., Otto M. J. Identification of a clinical isolate of HIV-1 with an isoleucine at position 82 of the protease which retains susceptibility to protease inhibitors. Antiviral Res. 1995 Sep;28(1):13–24. doi: 10.1016/0166-3542(95)00033-i. [DOI] [PubMed] [Google Scholar]
  16. Larder B. A., Kellam P., Kemp S. D. Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes. AIDS. 1991 Feb;5(2):137–144. doi: 10.1097/00002030-199102000-00002. [DOI] [PubMed] [Google Scholar]
  17. Lech W. J., Wang G., Yang Y. L., Chee Y., Dorman K., McCrae D., Lazzeroni L. C., Erickson J. W., Sinsheimer J. S., Kaplan A. H. In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects. J Virol. 1996 Mar;70(3):2038–2043. doi: 10.1128/jvi.70.3.2038-2043.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lin Y., Lin X., Hong L., Foundling S., Heinrikson R. L., Thaisrivongs S., Leelamanit W., Raterman D., Shah M., Dunn B. M. Effect of point mutations on the kinetics and the inhibition of human immunodeficiency virus type 1 protease: relationship to drug resistance. Biochemistry. 1995 Jan 31;34(4):1143–1152. doi: 10.1021/bi00004a007. [DOI] [PubMed] [Google Scholar]
  19. Markowitz M., Mo H., Kempf D. J., Norbeck D. W., Bhat T. N., Erickson J. W., Ho D. D. Selection and analysis of human immunodeficiency virus type 1 variants with increased resistance to ABT-538, a novel protease inhibitor. J Virol. 1995 Feb;69(2):701–706. doi: 10.1128/jvi.69.2.701-706.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Markowitz M., Saag M., Powderly W. G., Hurley A. M., Hsu A., Valdes J. M., Henry D., Sattler F., La Marca A., Leonard J. M. A preliminary study of ritonavir, an inhibitor of HIV-1 protease, to treat HIV-1 infection. N Engl J Med. 1995 Dec 7;333(23):1534–1539. doi: 10.1056/NEJM199512073332204. [DOI] [PubMed] [Google Scholar]
  21. Maschera B., Furfine E., Blair E. D. Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors. J Virol. 1995 Sep;69(9):5431–5436. doi: 10.1128/jvi.69.9.5431-5436.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Otto M. J., Garber S., Winslow D. L., Reid C. D., Aldrich P., Jadhav P. K., Patterson C. E., Hodge C. N., Cheng Y. S. In vitro isolation and identification of human immunodeficiency virus (HIV) variants with reduced sensitivity to C-2 symmetrical inhibitors of HIV type 1 protease. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7543–7547. doi: 10.1073/pnas.90.16.7543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Partaledis J. A., Yamaguchi K., Tisdale M., Blair E. E., Falcione C., Maschera B., Myers R. E., Pazhanisamy S., Futer O., Cullinan A. B. In vitro selection and characterization of human immunodeficiency virus type 1 (HIV-1) isolates with reduced sensitivity to hydroxyethylamino sulfonamide inhibitors of HIV-1 aspartyl protease. J Virol. 1995 Sep;69(9):5228–5235. doi: 10.1128/jvi.69.9.5228-5235.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Patick A. K., Mo H., Markowitz M., Appelt K., Wu B., Musick L., Kalish V., Kaldor S., Reich S., Ho D. Antiviral and resistance studies of AG1343, an orally bioavailable inhibitor of human immunodeficiency virus protease. Antimicrob Agents Chemother. 1996 Feb;40(2):292–297. doi: 10.1128/aac.40.2.292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Patick A. K., Rose R., Greytok J., Bechtold C. M., Hermsmeier M. A., Chen P. T., Barrish J. C., Zahler R., Colonno R. J., Lin P. F. Characterization of a human immunodeficiency virus type 1 variant with reduced sensitivity to an aminodiol protease inhibitor. J Virol. 1995 Apr;69(4):2148–2152. doi: 10.1128/jvi.69.4.2148-2152.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Richman D. D., Guatelli J. C., Grimes J., Tsiatis A., Gingeras T. Detection of mutations associated with zidovudine resistance in human immunodeficiency virus by use of the polymerase chain reaction. J Infect Dis. 1991 Dec;164(6):1075–1081. doi: 10.1093/infdis/164.6.1075. [DOI] [PubMed] [Google Scholar]
  27. Rose R. E., Gong Y. F., Greytok J. A., Bechtold C. M., Terry B. J., Robinson B. S., Alam M., Colonno R. J., Lin P. F. Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1648–1653. doi: 10.1073/pnas.93.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sardana V. V., Schlabach A. J., Graham P., Bush B. L., Condra J. H., Culberson J. C., Gotlib L., Graham D. J., Kohl N. E., LaFemina R. L. Human immunodeficiency virus type 1 protease inhibitors: evaluation of resistance engendered by amino acid substitutions in the enzyme's substrate binding site. Biochemistry. 1994 Mar 1;33(8):2004–2010. doi: 10.1021/bi00174a005. [DOI] [PubMed] [Google Scholar]
  29. Swanstrom R. Characterization of HIV-1 protease mutants: random, directed, selected. Curr Opin Biotechnol. 1994 Aug;5(4):409–413. doi: 10.1016/0958-1669(94)90050-7. [DOI] [PubMed] [Google Scholar]
  30. Tisdale M., Myers R. E., Maschera B., Parry N. R., Oliver N. M., Blair E. D. Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors. Antimicrob Agents Chemother. 1995 Aug;39(8):1704–1710. doi: 10.1128/aac.39.8.1704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Winslow D. L., Stack S., King R., Scarnati H., Bincsik A., Otto M. J. Limited sequence diversity of the HIV type 1 protease gene from clinical isolates and in vitro susceptibility to HIV protease inhibitors. AIDS Res Hum Retroviruses. 1995 Jan;11(1):107–113. doi: 10.1089/aid.1995.11.107. [DOI] [PubMed] [Google Scholar]
  32. Yamaguchi K., Byrn R. A. Clinical isolates of HIV-1 contain few pre-existing proteinase inhibitor resistance-conferring mutations. Biochim Biophys Acta. 1995 Dec 6;1253(2):136–140. doi: 10.1016/0167-4838(95)00167-1. [DOI] [PubMed] [Google Scholar]
  33. Zhang Y. M., Dawson S. C., Landsman D., Lane H. C., Salzman N. P. Persistence of four related human immunodeficiency virus subtypes during the course of zidovudine therapy: relationship between virion RNA and proviral DNA. J Virol. 1994 Jan;68(1):425–432. doi: 10.1128/jvi.68.1.425-432.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. el-Farrash M. A., Kuroda M. J., Kitazaki T., Masuda T., Kato K., Hatanaka M., Harada S. Generation and characterization of a human immunodeficiency virus type 1 (HIV-1) mutant resistant to an HIV-1 protease inhibitor. J Virol. 1994 Jan;68(1):233–239. doi: 10.1128/jvi.68.1.233-239.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES