alexa Nucleic acid template and the risk of a PCR-Induced HIV-1 drug resistance mutation.
Infectious Diseases

Infectious Diseases

Journal of AIDS & Clinical Research

Author(s): Varghese V, Wang E, Babrzadeh F, Bachmann MH, Shahriar R, , Varghese V, Wang E, Babrzadeh F, Bachmann MH, Shahriar R,

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Abstract BACKGROUND: The HIV-1 nucleoside RT inhibitor (NRTI)-resistance mutation, K65R confers intermediate to high-level resistance to the NRTIs abacavir, didanosine, emtricitabine, lamivudine, and tenofovir; and low-level resistance to stavudine. Several lines of evidence suggest that K65R is more common in HIV-1 subtype C than subtype B viruses. METHODS AND FINDINGS: We performed ultra-deep pyrosequencing (UDPS) and clonal dideoxynucleotide sequencing of plasma virus samples to assess the prevalence of minority K65R variants in subtype B and C viruses from untreated individuals. Although UDPS of plasma samples from 18 subtype C and 27 subtype B viruses showed that a higher proportion of subtype C viruses contain K65R (1.04\% vs. 0.25\%; p<0.001), limiting dilution clonal sequencing failed to corroborate its presence in two of the samples in which K65R was present in >1.5\% of UDPS reads. We therefore performed UDPS on clones and site-directed mutants containing subtype B- and C-specific patterns of silent mutations in the conserved KKK motif encompassing RT codons 64 to 66 and found that subtype-specific nucleotide differences were responsible for increased PCR-induced K65R mutation in subtype C viruses. CONCLUSIONS: This study shows that the RT KKK nucleotide template in subtype C viruses can lead to the spurious detection of K65R by highly sensitive PCR-dependent sequencing techniques. However, the study is also consistent with the subtype C nucleotide template being inherently responsible for increased polymerization-induced K65R mutations in vivo.
This article was published in PLoS One and referenced in Journal of AIDS & Clinical Research

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