Zidovudine (AZT) Monotherapy Selects for the A360V Mutation in the Connection Domain of HIV-1 Reverse Transcriptase

Background: We previously demonstrated in vitro that zidovudine (AZT) selects for A371V in the connection domain and Q509L in ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) which, together with the thymidine analog mutations D67N, K70R and T215F, confer greater than 100-fold AZT resistance. The goal of the current study was to determine whether AZT monotherapy in HIV-1 infected patients also selects the A371V, Q509L or other mutations in the C-terminal domains of HIV-1 RT. Methodology/Principal Findings: Full-length RT sequences in plasma obtained pre- and post-therapy were compared in 23 participants who received AZT monotherapy from the AIDS Clinical Trials Group study 175. Five of the 23 participants reached a primary study endpoint. Mutations significantly associated with AZT monotherapy included K70R (p = 0.003) and T215Y (p = 0.013) in the polymerase domain of HIV-1 RT, and A360V (p = 0.041) in the connection domain of HIV-1 RT. HIV-1 drug susceptibility assays demonstrated that A360V, either alone or in combination with thymidine analog mutations, decreased AZT susceptibility in recombinant viruses containing participant-derived full-length RT sequences or site-directed mutant RT. Biochemical studies revealed that A360V enhances the AZT-monophosphate excision activity of purified RT by significantly decreasing the frequency of secondary RNase H cleavage events that reduce the RNA/DNA duplex length and promote template/primer dissociation. Conclusions: The A360V mutation in the connection domain of RT was selected in HIV-infected individuals that received AZT monotherapy and contributed to AZT resistance. © 2012 Brehm et al

The K65R Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Exhibits Bidirectional Phenotypic Antagonism with Thymidine Analog Mutations

The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in vitro by many d-nucleoside analog RT inhibitors (NRTI) but has been rarely detected in treated patients. In recent clinical trials, the K65R mutation has emerged frequently in patients experiencing virologic failure on antiretroviral combinations that do not include 3′-azidothymidine (AZT). The reason for this change is uncertain. To gain insight, we examined trends in the frequency of K65R in a large genotype database, the association of K65R with thymidine analog mutations (TAMs) and other NRTI mutations, and the viral susceptibility profile of HIV-1 with K65R alone and in combination with TAMs. Among >60,000 clinical samples submitted for genotype analysis that contained one or more NRTI resistance mutations, the frequency of K65R increased from 0.4% in 1998 to 3.6% in 2003. Among samples with K65R, a strong negative association was evident with the TAMs M41L, D67N, L210W, T215Y/F, and K219Q/E (P < 0.005) but not with other NRTI mutations, including the Q151M complex. This suggested that K65R and TAMs are antagonistic. To test this possibility, we generated recombinant HIV-1 encoding K65R in two different TAM backgrounds: M41L/L210W/T215Y and D67N/K70R/T215F/K219Q. K65R reduced AZT resistance from >50-fold to <2.5-fold in both backgrounds. In addition, TAMs antagonized the phenotypic effect of K65R, reducing resistance to tenofovir, abacavir, 2′,3′-dideoxycytidine, dideoxyinosine, and stavudine. In conclusion, K65R and TAMs exhibit bidirectional phenotypic antagonism. This antagonism likely explains the negative association of these mutations in genotype databases, the rare emergence of K65R with antiretroviral therapies that contain AZT, and its more frequent emergence with combinations that exclude AZT

In Vitro Activity of Structurally Diverse Nucleoside Analogs against Human Immunodeficiency Virus Type 1 with the K65R Mutation in Reverse Transcriptase

Human immunodeficiency virus type 1 (HIV-1) with a lysine-to-arginine substitution at codon 65 (HIV-1(65R)) of reverse transcriptase (RT) can rapidly emerge in patients being treated with specific combinations of nucleoside analog RT inhibitors (NRTIs). A better understanding of the activity of approved and investigational NRTIs against HIV-1(65R) is needed to select optimal therapy for patients infected with this mutant and to devise strategies to prevent its emergence. Therefore, we tested a broad panel of NRTIs that differed by enantiomer, pseudosugar, and base component against HIV-1(65R) to determine how NRTI structure affects activity. Drug susceptibilities of recombinant wild-type (HIV-1(65K)) or mutant HIV-1(65R) were determined using a single-replication-cycle susceptibility assay with P4/R5 cells and/or a multiple-replication-cycle susceptibility assay with MT-2 cells. All d, l, and acyclic NRTIs were significantly less active against HIV-1(65R) than against HIV-1(65K) except for analogs containing a 3′-azido moiety. Pseudosugar structure and base component but not enantiomer influenced NRTI activity against HIV-1(65R). These findings support the inclusion of 3′-azido-3′-deoxythymidine in drug combinations to treat patients having HIV-1(65R) and to prevent its emergence

Zidovudine (AZT) Monotherapy Selects for the A360V Mutation in the Connection Domain of HIV-1 Reverse Transcriptase. PLoS One 2012; 7:e31558. at Pennsylvania State U niversity on M arch 4, 2016 http://cid.oxfordjournals.org/ D ow nloaded from Ac ce pt ed

Abstract Background: We previously demonstrated in vitro that zidovudine (AZT) selects for A371V in the connection domain and Q509L in ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) which, together with the thymidine analog mutations D67N, K70R and T215F, confer greater than 100-fold AZT resistance. The goal of the current study was to determine whether AZT monotherapy in HIV-1 infected patients also selects the A371V, Q509L or other mutations in the Cterminal domains of HIV-1 RT

AZT susceptibility of site-directed mutant HIV-1.

a<p>Wildtype (WT) is xxHIV-1_LAI.</p>b<p>Mean ± standard deviation from 4–6 independent experiments.</p>c<p>Average fold-resistance (Fold-R) of site-directed mutant EC₅₀ versus wildtype (WT).</p>d<p>Calculated using means of log₁₀ transformed EC₅₀ values and two-sided Student's <i>t</i> test.</p>e<p>Average Fold-R of 360 V versus A360 recombinant virus EC₅₀.</p>f<p>Calculated using means of log₁₀ transformed EC₅₀ values and two-sided Student's <i>t</i> test.</p

AZT susceptibility of recombinant viruses containing participant-derived RT sequences.

a<p>Mean ± standard deviation from 3–11 independent experiments.</p>b<p>Fold-resistance calculated by dividing EC₅₀ of mutant virus by EC₅₀ of wildtype (WT).</p>c<p>Calculated using means of log₁₀ transformed EC₅₀ values and two-sided Student's <i>t</i> test.</p>d<p>Fold-resistance calculated by dividing EC₅₀ of 360 V virus by EC₅₀ of 360 A virus.</p>e<p>Calculated using means of log₁₀ transformed EC₅₀ values and two-sided Student's <i>t</i> test.</p>f<p>Wildtype is xxLAI 3D (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031558#s4" target="_blank">Methods</a>).</p

Mutations selected by AZT monotherapy.

a<p>Two-sided McNemar's exact test between pre-therapy and AZT-experienced (N = 23 pairs). Not corrected for multiple comparisons.</p>b<p>TAMs listed in the IAS-USA 2010 drug resistance tables.</p