Gln145Met/Leu Changes in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Resistance to Nucleoside and Nonnucleoside Analogs and Impair Virus Replication

ABSTRACT The frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene in strains from a group of 3,595 highly active antiretroviral therapy (HAART)-experienced patients were 0.22, 2.36, and 0.86%, respectively. Several amino acid substitutions different from the recently reported Gln145Met change (S. Paolucci, F. Baldanti, M. Tinelli, G. Maga, and G. Gerna, AIDS 17:924-927, 2003) were detected at position 145. Thus, amino acid substitutions selected at position 145 were introduced into the wild-type HIV type 1 (HIV-1) RT gene by site-directed mutagenesis, and recombinant HIV strains were assayed for their drug susceptibilities. Only Met and Leu substitutions at position 145 of the HIV-1 RT conferred multidrug resistance, while other amino acid changes did not. Lower levels of replication of the Gln145Met recombinant strain compared with those of both Gln151Met and wild-type recombinant strains were observed. In in vitro inhibition assays, expression and purification of the recombinant Gln145Met HIV-1 RT revealed a strong loss of catalytic efficiency of the mutated enzyme, as well as significant resistance to both zidovudine and efavirenz. Specific amino acid substitutions in the HIV RT nucleotide-binding pocket might affect both antiretroviral drug recognition and binding and decrease the level of virus replication, possibly by interfering with the enzyme activity. This finding may explain the lower frequency of Gln145Met/Leu mutations observed compared with the frequencies of Gln151Met/Leu mutations and the insertion at position 69 in HAART-experienced patients.

[1]  Bikram Chakraborty,et al.  Insertions in the Reverse Transcriptase Increase both Drug Resistance and Viral Fitness in a Human Immunodeficiency Virus Type 1 Isolate Harboring the Multi-Nucleoside Reverse Transcriptase Inhibitor Resistance 69 Insertion Complex Mutation , 2002, Journal of Virology.

[2]  E. De Clercq.  HIV inhibitors targeted at the reverse transcriptase. , 1992, AIDS research and human retroviruses.

[3]  F. Maldarelli.  HIV-1 fitness and replication capacity: What are they and can they help in patient management? , 2003, Current infectious disease reports.

[4]  U Hübscher,et al.  Resistance to nevirapine of HIV-1 reverse transcriptase mutants: loss of stabilizing interactions and thermodynamic or steric barriers are induced by different single amino acid substitutions. , 1997, Journal of molecular biology.

[5]  J. Fantini,et al.  Multidrug resistance genotypes (insertions in the beta3-beta4 finger subdomain and MDR mutations) of HIV-1 reverse transcriptase from extensively treated patients: incidence and association with other resistance mutations. , 2000, Virology.

[6]  E. Arnold,et al.  Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  B Clotet,et al.  Antiretroviral drug resistance testing in adults with HIV infection: implications for clinical management. International AIDS Society--USA Panel. , 1998, JAMA.

[8]  M. Clementi,et al.  Dynamics of molecular parameters of human immunodeficiency virus type 1 activity in vivo , 1994, Journal of virology.

[9]  H. Mitsuya,et al.  Comparative Fitness of Multi-Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) in an In Vitro Competitive HIV-1 Replication Assay , 1999, Journal of Virology.

[10]  Dynamics of the Pool of Infected Resting Cd4 Hla-Dr- T Lymphocytes in Patients Who Started a Triple Class Five-Drug Antiretroviral Regimen During Primary HIV-1 Infection , 2003, Antiviral therapy.

[11]  N. Sluis-Cremer,et al.  Molecular mechanisms of HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs) , 2000, Cellular and Molecular Life Sciences (CMLS).

[12]  S. Deeks.  Nonnucleoside Reverse Transcriptase Inhibitor Resistance , 2001 .

[13]  D. Katzenstein,et al.  A 6-basepair insert in the reverse transcriptase gene of human immunodeficiency virus type 1 confers resistance to multiple nucleoside inhibitors. , 1998, The Journal of clinical investigation.

[14]  T. Merigan,et al.  Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy , 1996, Journal of virology.

[15]  F. Baldanti,et al.  Nevirapine-selected mutations Y181I/C of HIV-1 reverse transcriptase confer cross-resistance to stavudine , 2003, AIDS.

[16]  S. Hammer,et al.  The Relation between Baseline HIV Drug Resistance and Response to Antiretroviral Therapy: Re-Analysis of Retrospective and Prospective Studies Using a Standardized Data Analysis Plan , 2000, Antiviral therapy.

[17]  D. Stuart,et al.  A Family of Insertion Mutations between Codons 67 and 70 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confer Multinucleoside Analog Resistance , 1999, Antimicrobial Agents and Chemotherapy.

[18]  F. Baldanti,et al.  Novel recombinant phenotypic assay for clonal analysis of reverse transcriptase mutations conferring drug resistance to HIV-1 variants. , 2004, The Journal of antimicrobial chemotherapy.

[19]  D. Venzon,et al.  Altered drug sensitivity, fitness, and evolution of human immunodeficiency virus type 1 with pol gene mutations conferring multi-dideoxynucleoside resistance. , 1998, The Journal of infectious diseases.

[20]  F. Baldanti,et al.  Detection of a new HIV-1 reverse transcriptase mutation (Q145M) conferring resistance to nucleoside and non-nucleoside inhibitors in a patient failing highly active antiretroviral therapy. , 2003, AIDS.

[21]  P. Boyer,et al.  Subunit specificity of mutations that confer resistance to nonnucleoside inhibitors in human immunodeficiency virus type 1 reverse transcriptase , 1994, Antimicrobial Agents and Chemotherapy.

[22]  R. Shafer,et al.  Human immunodeficiency virus type 1 reverse-transcriptase and protease subtypes: classification, amino acid mutation patterns, and prevalence in a northern California clinic-based population. , 2001, The Journal of infectious diseases.

[23]  E. De Clercq,et al.  Prevalence and Characteristics of Multinucleoside-Resistant Human Immunodeficiency Virus Type 1 among European Patients Receiving Combinations of Nucleoside Analogues , 2000, Antimicrobial Agents and Chemotherapy.

[24]  R. Higuchi.  22 – RECOMBINANT PCR , 1990 .

[25]  F. Baldanti,et al.  Nevirapine Resistance Mutation at Codon 181 of the HIV-1 Reverse Transcriptase Confers Stavudine Resistance by Increasing Nucleotide Substrate Discrimination and Phosphorolytic Activity* , 2003, The Journal of Biological Chemistry.

[26]  N. Graham,et al.  Thymidine analog and multinucleoside resistance mutations are associated with decreased phenotypic susceptibility to stavudine in HIV type 1 isolated from zidovudine-naive patients experiencing viremia on stavudine-containing regimens. , 2001, AIDS research and human retroviruses.

[27]  L. Mansky.  Mutagenic outcome of combined antiviral drug treatment during human immunodeficiency virus type 1 replication. , 2003, Virology.

[28]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[29]  C. Petropoulos,et al.  Comparison of genotyping and phenotyping methods for determining susceptibility of HIV-1 to antiretroviral drugs , 2001, AIDS.