Mutations at Amino Acid Positions 63, 189, and 396 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Partially Restore the DNA Polymerase Activity of a TRP229TYR Mutant RT

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed “random scanning mutagenesis” of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.

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Convergent Evolution of Reverse Transcriptase (RT) Genes of Human Immunodeficiency Virus Type 1 Subtypes E and B following Nucleoside Analogue RT Inhibitor Therapies

Journal of Virology ◽

10.1128/jvi.74.11.5357-5362.2000 ◽

2000 ◽

Vol 74 (11) ◽

pp. 5357-5362 ◽

Cited By ~ 7

Author(s):

Hironori Sato ◽

Yasuhiro Tomita ◽

Kayo Shibamura ◽

Teiichiro Shiino ◽

Tuyoshi Miyakuni ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Nucleoside Analogue ◽

Subtype B ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Changes in the drug susceptibility, gene lineage, and deduced amino acid sequences of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) subtype E following 3′-azido-3′-deoxythymidine (AZT) monotherapy or AZT–2′,3′-dideoxyinosine combination therapy were examined with sequential virus isolates from a single family. The changes were compared to those reported for HIV-1 subtype B, revealing striking similarities in selected phenotype and amino acids independent of differences in the RT backbone sequences that constantly distinguish the two subtypes. Particularly, identical amino acid substitutions were present simultaneously at four different positions (D67N, K70R, T215F, and K219Q) for high-level AZT resistance. These data suggest that HIV-1 subtypes E and B evolve convergently at the phenotypic and amino acid levels when the nucleoside analogue RT inhibitors act as selective forces.

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Amino Acid Substitutions at Position 190 of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Increase Susceptibility to Delavirdine and Impair Virus Replication

Journal of Virology ◽

10.1128/jvi.77.2.1512-1523.2003 ◽

2003 ◽

Vol 77 (2) ◽

pp. 1512-1523 ◽

Cited By ~ 71

Author(s):

Wei Huang ◽

Andrea Gamarnik ◽

Kay Limoli ◽

Christos J. Petropoulos ◽

Jeannette M. Whitcomb

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Antiretroviral Drugs ◽

Replication Capacity ◽

Amino Acid Substitutions ◽

Immunodeficiency Virus

ABSTRACT Suboptimal treatment of human immunodeficiency virus type 1 (HIV-1) infection with nonnucleoside reverse transcriptase inhibitors (NNRTI) often results in the rapid selection of drug-resistant virus. Several amino acid substitutions at position 190 of reverse transcriptase (RT) have been associated with reduced susceptibility to the NNRTI, especially nevirapine (NVP) and efavirenz (EFV). In the present study, the effects of various 190 substitutions observed in viruses obtained from NNRTI-experienced patients were characterized with patient-derived HIV isolates and confirmed with a panel of isogenic viruses. Compared to wild-type HIV, which has a glycine at position 190 (G190), viruses with 190 substitutions (A, C, Q, S, V, E, or T, collectively referred to as G190X substitutions) were markedly less susceptible to NVP and EFV. In contrast, delavirdine (DLV) susceptibility of these G190X viruses increased from 3 to 300-fold (hypersusceptible) or was only slightly decreased. The replication capacity of viruses with certain 190 substitutions (C, Q, V, T, and E) was severely impaired and was correlated with reduced virion-associated RT activity and incomplete protease (PR) processing of the viral p55 gag polyprotein. These defects were the result of inadequate p160 gagpol incorporation into virions. Compensatory mutations within RT and PR improved replication capacity, p55 gag processing, and RT activity, presumably through increased incorporation of p160 gagpol into virions. We observe an inverse relationship between the degree of NVP and EFV resistance and the impairment of viral replication in viruses with substitutions at 190 in RT. These observations may have important implications for the future design and development of antiretroviral drugs that restrict the outgrowth of resistant variants with high replication capacity.

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