K65R in Subtype C HIV-1 Isolates from Patients Failing on a First-Line Regimen Including d4T or AZT: Comparison of Sanger and UDP Sequencing Data

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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

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

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

Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

Background: The impact of the products of the pol gene, specifically, reverse transcriptase (RT) on HIV-1 replication, evolution, and acquisition of drug resistance has been thoroughly characterized for subtype B. For subtype C, which accounts of almost 60% of HIV cases worldwide, much less is known. It has been reported that subtype C HIV-1 isolates have a lower replication capacity than B; however, the basis of these differences remains unclear. Results: We analyzed the impact of the pol gene products from HIV-1 B and C subtypes on the maturation of HIV virions, accumulation of reverse transcription products, integration of viral DNA, frequency of point mutations in provirus and overall viral replication. Recombinant HIV-1 viruses of B and C subtypes comprising the pol fragments encoding protease, integrase and either the whole RT or a chimeric RT from different isolates of the C and B subtypes, were used for infection of cells expressing CXCR4 or CCR5 co-receptors. The viruses carrying different fragments of pol from the isolates of B and C subtypes did not reveal differences in Gag and GagPol processing and viral RNA incorporation into the virions. However, the presence of the whole RT from subtype C, or the chimeric RT containing either the polymerase or the connection and RNase H domains from C isolates, caused significantly slower viral replication regardless of B or C viral backbone. Subtype C RT carrying viruses displayed lower levels of accumulation of strong-stop cDNA in permeabilized virions during endogenous reverse transcription, and decreased accumulation of both strong-stop and positive strand reverse transcription products in infected cells and in isolated reverse transcription complexes. This decreased accumulation correlated with lower levels of viral DNA integration in cells infected with viruses carrying the whole RT or RT domains from subtype C isolates. The single viral genome assay analysis did not reveal significant differences in the frequency of point mutations between the RT from B or C subtypes. Conclusions: These data suggest that the whole RT as well as distinct polymerase and connection-RNase H domains from subtype C HIV-1 confer a lower level of accumulation of reverse transcripts in the virions and reverse transcription complexes as compared to subtype B, resulting in a lower overall level of virus replication

Intersubtype differences in the effect of a rare p24 Gag mutation on HIV-1 replicative fitness.

Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P<0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness

Characterizing the emergence and persistence of drug resistant mutations in HIV-1 subtype C infections using 454 ultra deep pyrosequencing.

BACKGROUND: The role of HIV-1 RNA in the emergence of resistance to antiretroviral therapies (ARTs) is well documented while less is known about the role of historical viruses stored in the proviral DNA. The primary focus of this work was to characterize the genetic diversity and evolution of HIV drug resistant variants in an individual's provirus during antiretroviral therapy using next generation sequencing. METHODS: Blood samples were collected prior to antiretroviral therapy exposure and during the course of treatment from five patients in whom drug resistance mutations had previously been identified using consensus sequencing. The spectrum of viral variants present in the provirus at each sampling time-point were characterized using 454 pyrosequencing from multiple combined PCR products. The prevalence of viral variants containing drug resistant mutations (DRMs) was characterized at each time-point. RESULTS: Low abundance drug resistant viruses were identified in 14 of 15 sampling time-points from the five patients. In all individuals DRMs against current therapy were identified at one or more of the sampling time-points. In two of the five individuals studied these DRMs were present prior to treatment exposure and were present at high prevalence within the amplified and sequenced viral population. DRMs to drugs other than those being currently used were identified in four of the five individuals. CONCLUSION: The presence of DRMs in the provirus, regardless of their observed prevalence did not appear to have an effect on clinical outcomes in the short term suggesting that the drug resistant viral variants present in the proviral DNA do not appear to play a role in the short term in facilitating the emergence of drug resistance

Structural Basis for the Role of the K65R Mutation in HIV-1 Reverse Transcriptase Polymerization, Excision Antagonism, and Tenofovir Resistance*

K65R is a primary reverse transcriptase (RT) mutation selected in human immunodeficiency virus type 1-infected patients taking antiretroviral regimens containing tenofovir disoproxil fumarate or other nucleoside analog RT drugs. We determined the crystal structures of K65R mutant RT cross-linked to double-stranded DNA and in complexes with tenofovir diphosphate (TFV-DP) or dATP. The crystals permit substitution of TFV-DP with dATP at the dNTP-binding site. The guanidinium planes of the arginines K65R and Arg72 were stacked to form a molecular platform that restricts the conformational adaptability of both of the residues, which explains the negative effects of the K65R mutation on nucleotide incorporation and on excision. Furthermore, the guanidinium planes of K65R and Arg72 were stacked in two different rotameric conformations in TFV-DP- and dATP-bound structures that may help explain how K65R RT discriminates the drug from substrates. These K65R-mediated effects on RT structure and function help us to visualize the complex interaction with other key nucleotide RT drug resistance mutations, such as M184V, L74V, and thymidine analog resistance mutations