Template Usage Is Responsible for the Preferential Acquisition of the K65R Reverse Transcriptase Mutation in Subtype C Variants of Human Immunodeficiency Virus Type 1
ABSTRACT 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.
Full-textDOI: · Available from: Daniela Moisi, Jun 24, 2014
- SourceAvailable from: Juan Vicente Guanira
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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). "
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.The Journal of Infectious Diseases 04/2014; 210(8). DOI:10.1093/infdis/jiu233 · 6.00 Impact Factor
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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  , whereas the subtype B template favors pausing at codon 67 that may facilitate the generation of D67N and TAMs rather than K65R   . 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   . Figure 1 provides a pictorial representation of the preferential development of K65R in subtype C viruses. "
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.06/2012; 2012(4):256982. DOI:10.1155/2012/256982
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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 –. Moreover, it has also been shown that the KKK motif in this subtype can lead to PCR-induced K65R . "
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. 27 subtype C HIV-1 isolates from treated patients failing on a regimen with d4T or AZT plus lamivudine (3TC) plus nevirapine (NVP) or efavirenz (EFV) and who had been sequenced by Sanger were investigated by UDPS at codon 65 of the reverse transcriptase (RT). 18 isolates from naïve patients and dilutions of a control K65R plasmid were analysed by Sanger plus UDPS. Analysis of Sanger sequences of subtype C HIV-1 isolates from naïve patients exhibited expected polymorphic substitutions compared to subtype B but no drug resistance mutations (DRMs). Quantitation of K65R variants by UDPS ranged from <0.4% to 3.08%. Sanger sequences of viral isolates from patients at failure of d4T or AZT plus 3TC plus NVP or EFV showed numerous DRMs to nucleoside reverse transcriptase inhibitors (NRTIs) including M184V, thymidine-associated mutations (TAMs) plus DRMs to non- nucleoside reverse transcriptase inhibitors (NNRTIs). Two K65R were observed by Sanger in this series of 27 samples with UDPS percentages of 27 and 87%. Other samples without K65R by Sanger exhibited quantities of K65R variants ranging from <0.4% to 0.80%, which were below the values observed in isolates from naïve patients. While Sanger sequencing of subtype C isolates from treated patients at failure of d4T or AZT plus 3TC plus NVP or EFV exhibited numerous mutations including TAMs and 8% K65R, UDPS quantitation of K65R variants in the same series did not provide any more information than Sanger.PLoS ONE 05/2012; 7(5):e36549. DOI:10.1371/journal.pone.0036549 · 3.23 Impact Factor