Nucleic Acid Template and the Risk of a PCR-Induced HIV-1 Drug Resistance Mutation
2010 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 5, no 6, e10992- p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2010. Vol. 5, no 6, e10992- p.
IdentifiersURN: urn:nbn:se:kth:diva-159917DOI: 10.1371/journal.pone.0010992ISI: 000278465900010PubMedID: 20539818ScopusID: 2-s2.0-77956204474OAI: oai:DiVA.org:kth-159917DiVA: diva2:787708
QC 201502112015-02-112015-02-112015-02-11Bibliographically approved