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The y271 and i274 amino acids in reverse transcriptase of human immunodeficiency virus-1 are critical to protein stability.

ABSTRACT: Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 plays a key role in initiating viral replication and is an important target for developing anti-HIV drugs. Our previous study showed that two mutations (Y271A and I274A) in the turn RT (Gln(269)-Arg(277)) abrogated viral replication, but the replication capacity and RT activity was discordant. In this study, we further investigated why alanine substitutions at these two sites would affect viral replication. We found that both RT activity and RT protein were almost undetectable in viral particles of these two mutants, although the Pr160(gag-pol) mutants were properly expressed, transported and incorporated. Using protease inhibition assay, we demonstrated a correlation between the degradation of the RT mutants and the activity of viral protease. Our native gel analysis indicated that the mutations at 271 and 274 amino acids might cause conformational changes, leading to the formation of higher order oligomers instead of dimers, resulting in increased protein instability and susceptibility to viral protease. Thus, residues 271 and 274 are critical to RT stability and resistance to viral protease. The conservation of the two amino acid residues among different strains of HIV-1 lent further support to this conclusion. The knowledge gained here may prove useful in drug design.

SUBMITTER: Zhang HJ

PROVIDER: S-EPMC2701634 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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The y271 and i274 amino acids in reverse transcriptase of human immunodeficiency virus-1 are critical to protein stability.

Zhang Hao-Jie HJ Wang Yong-Xiang YX Wu Hao H Jin Dong-Yan DY Wen Yu-Mei YM Zheng Bo-Jian BJ

PloS one 20090703 7

Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 plays a key role in initiating viral replication and is an important target for developing anti-HIV drugs. Our previous study showed that two mutations (Y271A and I274A) in the turn RT (Gln(269)-Arg(277)) abrogated viral replication, but the replication capacity and RT activity was discordant. In this study, we further investigated why alanine substitutions at these two sites would affect viral replication. We found that both RT ...[more]

PMID: 19578544

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Project description:The development of antiviral drug resistance is an important problem in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Potent antiretroviral therapy is currently used for treatment, and typically consists of at least two reverse transcriptase (RT) inhibitors. We have previously reported that both drugs and drug-resistant RT mutants can increase virus mutation frequencies. To further assess the contributions of nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and drug-resistant RTs to HIV mutagenesis, a new high-throughput assay system was developed. This assay system was designed to specifically detect frameshift mutations in the luciferase gene in a single virus replication cycle. New drug-resistant RTs were identified that significantly altered virus mutation frequencies. Consistent with our previous observations of NRTIs, abacavir, stavudine, and zalcitabine increased HIV-1 mutation frequencies, supporting the general hypothesis that the NRTIs currently used in antiviral drug therapy increase virus mutation frequencies. Interestingly, similar observations were made with NNRTIs. This is the first report to show that NNRTIs can influence virus mutation frequencies. NNRTI combinations, NRTI-NNRTI combinations, and combinations of drug and drug-resistant RTs led to significant changes in the virus mutation frequencies compared to virus replication of drug-resistant virus in the absence of drug or wild-type virus in the presence of drug. This indicates that combinations of RT drugs or drugs and drug-resistant virus created during the evolution of drug resistance can act together to increase HIV-1 mutation frequencies, which would have important implications for drug therapy regimens. Finally, the influence of drug-resistant RT mutants from CRF01_AE viruses on HIV-1 mutation frequencies was analyzed and it was found that only a highly drug resistant RT led to altered virus mutation frequencies. The results further suggest that high-level drug-resistant RT can significantly influence virus mutation frequencies. A structural model that explains the mutation frequency data is discussed.

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The y271 and i274 amino acids in reverse transcriptase of human immunodeficiency virus-1 are critical to protein stability.

Publications

The y271 and i274 amino acids in reverse transcriptase of human immunodeficiency virus-1 are critical to protein stability.

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