Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance

<p>Abstract</p> <p>Background</p> <p>We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT).</p> <p>Methods</p> <p>Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from <it>Escherichia coli</it>. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3^Lysas primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments.</p> <p>Results</p> <p>The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT.</p> <p>Conclusion</p> <p>The diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.</p

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

Mechanisms of drug resistance development in subtype B and C variants of human immunodeficiency virus type-1

The human immunodeficiency virus type-1 (HIV-1) can be phylogentically classified into multiple groups, subtypes, sub-subtypes and recombinant forms. Subtype B HIV-1 is responsible for approximately 10% of the pandemic and is primarily located in the developed regions of the world, whereas subtype C HIV-1 is responsible for over 50% of the pandemic and is primarily located in the sub-Saharan African countries. The majority of HIV-1 research to date has focused on subtype B HIV-1 and, as a result, little is known about subtype differences or about drug resistance development between the subtypes. Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C as opposed to subtype B HIV-1. We described that this discrepancy partly lies in the subtype C template coding sequence that predisposes RT to pause strongly at the site of K65R development. This pausing reaction was shown to be specific to the subtype C HIV-1 coding sequence in addition to being difficult to alleviate. We also showed that DNA synthesis assays performed with subtype C templates consistently produced more K65R-containing transcripts than with subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism 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 on the subtype C sequence. Thus, the data show that during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region, strand slippage can occur resulting in the 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 or, upon dislocation and realignment of the primer and template, the development of the K65R mutation. These findings provide specific mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1 and demonstrate the drug resistance mutations can develop differently between HIV-1 subtypes. A more complete understanding of the clinical implications of these findings requires the long-term follow up of patients on antiretroviral therapy in subtype C endemic regions of the world.Les virus de l'immunodéficience humain de type-1 (VIH-1) peuvent être classifiés en plusieurs groupes, sous-types, sous sous-types et formes recombinantes. Le VIH-1 de sous-type B est responsable d'environ 10% de la pandémie globale qui sévit principalement dans les pays développés du monde. Cependant, le VIH-1 de sous-type C est responsable pour plus de 50% de la pandémie qui se situe principalement dans les pays en voie de développement du monde y compris les pays dans le sud de l'Afrique. La majorité de la recherche sur le VIH-1 se fait sur des virus de sous-type B et, par conséquence, on en connait peu sur les différences entre les sous-types et sur le développement de résistance aux antirétroviraux entre ces derniers. Plusieurs études suggérèrent que la mutation K65R dans la transcriptase inverse (TI) apparait plus rapidement chez les virus de sous-type C que chez ceux de sous-type B. Nous avons établit que la matrice génétique dérivé des virus de sous-type C est, en partie, responsable pour ces observations puisqu'elle entraine un arrêt de la synthèse d'ADN par la polymérase au nucléotide spécifique responsable pour l'apparition de la mutation K65R. Cet arrêt semble spécifique à la matrice génétique des virus de sous-type C et peut être difficilement empêché. Nous avons également montré que des matrices de sous-type C produisent de façon constante des produits ayant la mutation K65R, et ce avec une plus grande fréquence que pour les matrices de sous-type B. Peu importe le sous-type des TI utilisées lors des expériences, les résultats demeurent les mêmes. Ces donnés suggèrent que le mécanisme responsable est spécifique de l'origine de la matrice mais indépendant de l'origine de la TI. De plus, la synthèse d'ADN se déroule d'une façon particulière qui suggère un glissement entre la matrice et l'amorce ce qui entraine une dislocation sur la matrice génétique de sous-type C. Nos résultats montrent que lors de la synthèse d'ADN à partir de séquences nucléotidiques homopolymériques se situant autour de la région K65 de sous-type C, un glissement entre les brins d'ADN peut entrainer un désalignement entre la matrice et l'amorce. Par la suite, ce glissement peut supprimer l'adénine au milieu du codon K65 et produire une mutation de changement de phase ou, suivant une dislocation avec réalignement de la matrice et de l'amorce, produire la mutation K65R. Ces résultats suggèrent que l'apparition de la mutation K65R chez le virus de sous-type C est facilitée et que l'émergence de mutations de résistance peut se produire de façon divergente entre les différents sous-types de VIH-1. Pour mieux comprendre les implications cliniques de ces observations, il faudrait un suivi à long terme des patients traités avec des antirétroviraux dans des régions du monde endémiques par le VIH-1 de sous-type C

Molecular characterization of the development of the K65R and M184V drug resistance mutations in Subtype C HIV-1s

Background: We have shown that the K65R mutation is selected more rapidly in subtype C than in subtype B HIV-1 isolates in both cell culture and clinical studies. Biochemical comparisons between subtype B and C-derived reverse transcriptase (RT) enzymes revealed similar molecular characteristics that do not explain the more rapid selection of K65R with subtype C viruses. This study attempts to establish the mechanistic basis for the difference. Methods: Recombinant subtype C and B HIV-1 RT enzymes were expressed and purified in E. coli. Gel-based nucleotide extension assays were used to study DNA synthesis from various natural and synthetic DNA and RNA templates that spanned regions of the pol gene responsible for the K65R and M184V mutations. Cell based experiments were performed using MT2 cells infected with mutated subtype B HIV-1 pNL4-3 viruses. Results: The propensity for the more rapid selection of K65R with subtype C enzymes is due to the mechanism of DNA synthesis from a subtype C template. The use of templates containing the 64, 65 and 66 codons of the pol gene led to different patterns of DNA synthesis. When subtype C RT was employed to synthesize DNA from subtype C templates, preferential pausing was seen at the nucleotide position responsible for the AAG to AGG mutation on codon 65 which gives rise to K65R. In contrast, the use of subtype B RT together with a subtype B template reveals a different pattern of DNA synthesis. When subtype B RT was employed with a subtype C template, DNA synthesis stopped at the exact nucleotide position responsible for K65R. This phenomenon was not observed when subtype C RT was used with a subtype B template. A similar method was employed to investigate if differences exist in the appearance of M184V between subtypes. The results suggest that M184V is not favoured due to its coding sequence and that the propensity for the development of M184V remains the same in subtype B and C HIV. In cell culture, K65R was detected faster in subtype B that has been mutated to include the 64/65 codons of subtype C, when compared to wild-type subtype B HIV. Conclusions: The more rapid emergence of K65R but not M184V in subtype C RT appears to be based on the pol gene coding sequence. These results urge for the analysis of resistance mechanisms to be studied in all HIV subtypes separately and have clinical relevance in regard to the management of subtype C infections