A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

Abstract

Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1.

Low-Abundance Resistant Mutations in HIV-1 Subtype C Antiretroviral Therapy-Naive Individuals as Revealed by Pyrosequencing

“…Additionally, low abundance of K65R have been detected by UDPS in ART-naïve non-B subtypes and correlated with virological failure [5]. The predominance of K65R in subtype C can be explained by a template pausing molecular mechanism that facilitates the selection of K65R in the KKK motif at codons 64, 65, 66 of the RT [30–32], and has been suggested that this characteristic of the KKK motif can lead to PCR induced K65R [33]. However, a recent UDPS study were controls of different percentages of K65R mutation were included and the theoretical and observed values of K65R plasmid dilutions were similar (100%:94%, 5%:2.30% and 1%:0.90%), indicates that the frequencies of K65R obtained by UDPS are reasonably accurate [34].…”

HIV-1 Genetic Variability and Clinical Implications

“…This leads to reverse transcriptase pausing during the synthesis of double-stranded DNA from the single-stranded DNA intermediate template, a process that is template-specific but independent of the reverse transcriptase enzyme [14, 15, 148]. Subsequent misalignment of the subtype C template and primer leads to the AAG to AGG change responsible for the K65R mutation [149] (Figure 2). On the contrary, subtype B templates are prone to frequent pausing at codon 67, facilitating the generation of mutation D67N and TAMs instead of K65R (Figure 2, Table 1) [148, 150, 151].…”

Next generation sequencing improves detection of drug resistance mutations in infants after PMTCT failure

“…Interestingly, NVP-containing regimens have been associated with an increased risk of K65R detection [27,28], but the mechanism has not yet been described. Overall, while HIV-1 subtype C has an increased risk of K65R under drug pressure[27,29], the extent to which these K65R mutations are from reverse transcription error in vitro or in vivo, is unclear.…”