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Silent mutations are selected in HIV-1 reverse transcriptase and affect enzymatic efficiency.

ABSTRACT: Missense mutations in HIV-1 reverse transcriptase are frequently selected in response to therapy; we examined whether silent mutations were also selected for by HIV therapy.Retrospective, observational analysis. Biochemical assays.A comparison of the reverse transcriptase gene, from antiretroviral- naive (N = 812) and experienced individuals (N = 2212), reveals two silent mutations (K65K and K66K) that are strongly associated with treatment experience. To assess reverse transcription efficiency, steady-state kinetic assays were carried out using recombinant purified HIV-1 reverse transcriptase and a series of synthetic RNA/DNA template/primer substrates. The RNA templates spanned codons 60-77 in the reverse transcriptase and included different combinations of mutations at codons 65, 66, 67, and 70.Silent AAG mutations (or mixtures) at reverse transcriptase codons 65 and/or 66 were observed in 812 samples from 351 patients and 2129 samples from 829 patients, respectively. In clade B samples, there was a very strong relationship between the silent mutations and the thymidine analogue mutations, in particular D67N. Steady-state kinetic experiments demonstrated that HIV-1 reverse transcriptase exhibited a strong tendency to pause and/or dissociate at codons 65 and 66 on RNA templates that contained the D67N and K70R mutations. However, when the K66 or K66 AAA to AAG mutations were added to the background of the 67 and 70 mutational changes, these pausing and/or dissociation events were largely alleviated.Silent mutations at codons 65 and/or 66 are strongly coselected with thymidine analogue mutations. These data provide the first evidence for an RNA-level mechanism of direct relevance to drug resistance.

SUBMITTER: Harrigan PR

PROVIDER: S-EPMC4829083 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Silent mutations are selected in HIV-1 reverse transcriptase and affect enzymatic efficiency.

Harrigan P Richard PR Sheen Chih-Wei CW Gill Vikram S VS Wynhoven Brian B Hudson Erin E Lima Viviane D VD Lecocq Pierre P Aguirre Rosalia R Poon Art F Y AF Sluis-Cremer Nicolas N

AIDS (London, England) 20081101 18

<h4>Objective</h4>Missense mutations in HIV-1 reverse transcriptase are frequently selected in response to therapy; we examined whether silent mutations were also selected for by HIV therapy.<h4>Design</h4>Retrospective, observational analysis. Biochemical assays.<h4>Methods</h4>A comparison of the reverse transcriptase gene, from antiretroviral- naive (N = 812) and experienced individuals (N = 2212), reveals two silent mutations (K65K and K66K) that are strongly associated with treatment experi ...[more]

PMID: 19005273

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Project description:UnlabelledThe genetic variation in HIV-1 in patients is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Some of the mutations in reverse transcriptase (RT) that alter the deoxynucleoside triphosphate (dNTP)-binding pocket, including those that confer resistance to nucleoside/nucleotide analogs, affect dNTP selection during replication. The effects of mutations in RT on the spectrum (nature, position, and frequency) of errors made in vivo are poorly understood. We previously determined the mutation rate and the frequency of different types of mutations and identified hot spots for mutations in a lacZα (the α complementing region of lacZ) reporter gene carried by an HIV-1 vector that replicates using wild-type RT. We show here that four mutations (Y115F, M184V, M184I, and Q151M) in the dNTP-binding pocket of RT that had relatively small effects on the overall HIV-1 mutation rate (less than 3-fold compared to the wild type) significantly increased mutations at some specific positions in the lacZα reporter gene. We also show that changes in a sequence that flanks the reporter gene can affect the mutations that arise in the reporter. These data show that changes either in HIV-1 RT or in the sequence of the nucleic acid template can affect the spectrum of mutations made during viral replication. This could, by implication, affect the generation of drug-resistant mutants and immunological-escape mutants in patients.ImportanceRT is the viral enzyme that converts the RNA genome of HIV into DNA. Errors made during replication allow the virus to escape from the host's immune system and to develop resistance to the available anti-HIV drugs. We show that four different mutations in RT which are known to be associated with resistance to anti-RT drugs modestly increased the overall frequency of errors made during viral replication. However, the increased errors were not uniformly distributed; the additional errors occurred at a small number of positions (hot spots). Moreover, some of the RT mutations preferentially affected the nature of the errors that were made (some RT mutations caused an increase in insertion and deletion errors; others caused an increase in substitution errors). We also show that sequence changes in a region adjacent to a target gene can affect the errors made within the target gene.

Dataset Information

Silent mutations are selected in HIV-1 reverse transcriptase and affect enzymatic efficiency.

Publications

Silent mutations are selected in HIV-1 reverse transcriptase and affect enzymatic efficiency.

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