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

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

ATP-mediated AZT-MP excision activity and RNase H activity of wildtype, A360V, TAM-1 and TAM-1/A360V HIV-1 RT.

<p><b>A</b>) Isotherms of ATP-mediated AZT-MP excision reactions carried out by wildtype and mutant HIV-1 RT on a DNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. Reaction times were: wildtype and A360V = 10, 20, 30, 45, 60, 75, 90, 105 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. <b>B</b>) Isotherms of ATP-mediated AZT-MP excision reactions carried out by wildtype and mutant HIV-1 RT on an RNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. Reaction times were: wildtype and A360V = 15, 30, 45, 60, 75, 90, 105, 120 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. <b>C</b>) Representative autoradiogram of the RNase H cleavage activity of the wildtype and mutant HIV-1 RTs. Experiments were carried out as described in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031558#s4" target="_blank">Materials and Methods</a></i>. The reaction times were wildtype and A360V = 15, 30, 45, 60, 75, 90, 105, 120 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. <b>D</b>) Isotherms for the accumulation of the −10 product formed by wildtype and mutant HIV-1 RT during AZT-MP excision.</p

Pattern of emergence of resistance mutations among participants who selected A360V.

a<p>Sample obtained at earliest available time point after treatment initiation and which mutations were present compared with the pre-therapy sample.</p>b<p>Sample obtained at time point of last-on-therapy sample available. Participants 3 and 4 reached a study endpoint. Participants 6, 11, 29 and 31 did not reach a study end-point.</p>c<p>For participants 6 and 11, mutation A360V occurred in <25% of the viral population which is difficult to identify by population sequencing.</p

Mutations and polymorphisms associated with A360V.

a<p>Mutations/polymorphisms of the viral population in each participant sample compared to consensus subtype B RT (Los Alamos HIV Sequence Database);</p>b<p>TAMs highlighted in bold;</p>c<p>Mutations/polymorphisms of an individual participant-derived recombinant clone when compared to consensus subtype B RT (Los Alamos HIV Sequence Database).</p

Anti-Human Immunodeficiency Virus Activity, Cross-Resistance, Cytotoxicity, and Intracellular Pharmacology of the 3′-Azido-2′,3′-Dideoxypurine Nucleosides▿

Although the approved nucleoside reverse transcriptase (RT) inhibitors (NRTI) are integral components of therapy for human immunodeficiency virus type 1 (HIV-1) infection, they can have significant limitations, including the selection of NRTI-resistant HIV-1 and cellular toxicity. Accordingly, there is a critical need to develop new NRTI that have excellent activity and safety profiles and exhibit little or no cross-resistance with existing drugs. In this study, we report that the 3′-azido-2′,3′-dideoxypurine nucleosides (ADPNs) 3′-azido-2′,3′-dideoxyadenosine (3′-azido-ddA) and 3′-azido-2′,3′-dideoxyguanosine (3′-azido-ddG) exert potent antiviral activity in primary human lymphocytes and HeLa and T-cell lines (50% inhibitory concentrations [IC50s] range from 0.19 to 2.1 μM for 3′-azido-ddG and from 0.36 to 10 μM for 3′-azido-ddA) and that their triphosphate forms are incorporated as efficiently as the natural dGTP or dATP substrates by HIV-1 RT. Importantly, both 3′-azido-ddA and 3′-azido-ddG retain activity against viruses containing K65R, L74V, or M184V (IC50 change of <2.0-fold) and against those containing three or more thymidine analog mutations (IC50 change of <3.5-fold). In addition, 3′-azido-ddG does not exhibit cytotoxicity in primary lymphocytes or epithelial or T-cell lines and does not decrease the mitochondrial DNA content of HepG2 cells. Furthermore, 3′-azido-ddG is efficiently phosphorylated to 3′-azido-ddGTP in human lymphocytes, with an intracellular half-life of the nucleoside triphosphate of 9 h. The present data suggest that additional preclinical studies are warranted to assess the potential of ADPNs for treatment of HIV-1 infection