Template Usage Is Responsible for the Preferential Acquisition of the K65R Reverse Transcriptase Mutation in Subtype C Variants of Human Immunodeficiency Virus Type 1

McGill University AIDS Center, McGill University, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Canada.
Journal of Virology (Impact Factor: 4.44). 02/2009; 83(4):2029-33. DOI: 10.1128/JVI.01349-08
Source: PubMed


We propose that a nucleotide template-based mechanism facilitates the acquisition of the K65R mutation in subtype C human
immunodeficiency virus type 1 (HIV-1). Different patterns of DNA synthesis were observed using DNA templates from viruses
of subtype B or C origin. When subtype C reverse transcriptase (RT) was employed to synthesize DNA from subtype C DNA templates,
preferential pausing was seen at the nucleotide position responsible for the AAG-to-AGG K65R mutation. This did not occur
when the subtype B RT and template were used. Template factors can therefore increase the probability of K65R development
in subtype C HIV-1.

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Available from: Daniela Moisi, Jun 24, 2014
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    • "Viral sequence diversity presents challenges for quantitative target sequence-based diagnostics, especially in homopolymer regions adjacent to DR codons such as subtype C K65R [29, 30, 32]. To increase the specificity and accuracy of AS-PCR strategies for minor variant quantification across multiple subtypes, we developed the quantitative minor variant assay (qMVA), which includes a “cure” PCR step before the AS-PCR to normalize the primer target sites adjacent to the SNP conferring resistance to defined, consensus-based sequences (Figure 2). "
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    ABSTRACT: Background: The iPrEx study demonstrated that combination oral emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) as preexposure prophylaxis (PrEP) protects against HIV acquisition in men who have sex with men and transgender women. Selection for drug resistance could offset PrEP benefits. Methods: Phenotypic and genotypic clinical resistance assays characterized major drug resistant mutations. Minor variants with FTC/TDF mutations K65R, K70E, M184V/I were measured using 454 deep sequencing and a novel allele-specific polymerase chain reaction (AS-PCR) diagnostic tolerant to sequence heterogeneity. Results: Control of primer-binding site heterogeneity resulted in improved accuracy of minor variant measurements by AS-PCR. Of the 48 on-study infections randomized to FTC/TDF, none showed FTC/TDF mutations by clinical assays despite detectable drug levels in 8 participants. Two randomized to FTC/TDF had minor variant M184I detected at 0.53% by AS-PCR or 0.75% by deep sequencing, only 1 of which had low but detectable drug levels. Among those with acute infection at randomization to FTC/TDF, M184V or I mutations that were predominant at seroconversion waned to background levels within 24 weeks after discontinuing drug. Conclusions: Drug resistance was rare in iPrEx on-study FTC/TDF-randomized seroconverters, and only as low-frequency minor variants. FTC resistance among those initiating PrEP with acute infection waned rapidly after drug discontinuation. Clinical Trials Registration.NCT00458393.
    Full-text · Article · Apr 2014 · The Journal of Infectious Diseases
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    • "Subtype C viruses apparently have an intrinsic difficulty in synthesizing stretches of adenine homopolymeric runs that leads to template pausing at codon 65, facilitating the acquisition of K65R under selective drug pressure [37] [38], whereas the subtype B template favors pausing at codon 67 that may facilitate the generation of D67N and TAMs rather than K65R [37] [38] [39]. In addition, the introduction of codons from positions 64 and 65 in the RT of subtype C into a subtype B backbone was sufficient to lead to selection of K65R by multiple NRTIs [37] [38] [39]. Figure 1 provides a pictorial representation of the preferential development of K65R in subtype C viruses. "
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    ABSTRACT: The vast majority of reports on drug resistance deal with subtype B infections in developed countries, and this is largely due to historical delays in access to antiretroviral therapy (ART) on a worldwide basis. This notwithstanding the concept that naturally occurring polymorphisms among different non-B subtypes can affect HIV-1 susceptibility to antiretroviral drugs (ARVs) is supported by both enzymatic and virological data. These findings suggest that such polymorphisms can affect both the magnitude of resistance conferred by some major mutations as well as the propensity to acquire certain resistance mutations, even though such differences are sometimes difficult to demonstrate in phenotypic assays. It is mandatory that tools are optimized to assure accurate measurements of drug susceptibility in non-B subtypes and to recognize that each subtype may have a distinct resistance profile and that differences in resistance pathways may also impact on cross-resistance and the choice of regimens to be used in second-line therapy. Although responsiveness to first-line therapy should not theoretically be affected by considerations of viral subtype and drug resistance, well-designed long-term longitudinal studies involving patients infected by viruses of different subtypes should be carried out.
    Full-text · Article · Jun 2012
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    • "From a molecular point of view, it has been demonstrated that the RT KKK nucleotide motif at codons 64, 65, 66 in reverse transcriptase of subtype C HIV-1 appears to lead to template pausing that facilitates the selection of K65R, even in isolates from untreated patients [9]–[12]. Moreover, it has also been shown that the KKK motif in this subtype can lead to PCR-induced K65R [13]. "
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    ABSTRACT: We and others have shown that subtype C HIV-1 isolates from patients failing on a regimen containing stavudine (d4T) or zidovudine (AZT) exhibit thymidine-associated mutations (TAMs) and K65R which can impair the efficacy of Tenofovir (TDF) at second line. Depending on the various studies, the prevalence of K65R substitution as determined by the Sanger method ranges from 4 to 30%. Our aim was to determine whether ultra-deep pyrosequencing (UDPS) could provide more information than the Sanger method about selection of K65R in this population of patients.
    Full-text · Article · May 2012 · PLoS ONE
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