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

Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo

Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2–9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow–liver–thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal—in combination with appropriate tools for systemic clearance of persistent HIV infection—greatly increases opportunities for HIV eradication

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

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

Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4+ T Cells.

During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal.IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells

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

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