Dianna Koontz

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

ABSTRACT 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.

Selection of Mutations in the Connection and RNase H Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Increase Resistance to 3′-Azido-3′-Dideoxythymidine

ABSTRACT Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3′-azido-3′-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1LAI or AZT-resistant HIV-1LAI (M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1LAI were two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance (∼16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance ∼10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.

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

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) with a lysine-to-arginine substitution at codon 65 (HIV-165R) 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-165R 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-165R to determine how NRTI structure affects activity. Drug susceptibilities of recombinant wild-type (HIV-165K) or mutant HIV-165R 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-165R than against HIV-165K except for analogs containing a 3′-azido moiety. Pseudosugar structure and base component but not enantiomer influenced NRTI activity against HIV-165R. These findings support the inclusion of 3′-azido-3′-deoxythymidine in drug combinations to treat patients having HIV-165R and to prevent its emergence.

The 3'-azido group is not the primary determinant of 3'-azido-3'-deoxythymidine (AZT) responsible for the excision phenotype of AZT-resistant HIV-1

The mechanism of human immunodeficiency virus (HIV) 1 resistance to 3′-azido-3′-deoxythymidine (AZT) involves reverse transcriptase (RT)-catalyzed phosphorolytic excision of the chain-terminating AZT-5′-monophosphate (AZTMP). Primers terminated with AZTMP are generally better substrates for this reaction than those terminated with 2′,3′-dideoxynucleoside-5′-monophosphate (2′,3′-ddNMP) analogs that lack a 3′-azido moiety. This led to the hypothesis that the 3′-azido group is a major structural determinant for maintaining the primer terminus in the appropriate site for phosphorolytic excision of AZTMP by AZT-resistant (AZTR) RT. To test this hypothesis, we evaluated the incorporation, phosphorolytic excision, and antiviral activity of a panel of 3′-azido-2′,3′-ddN including 3′-azido-2′,3′-ddA (AZddA), 3′-azido-2′,3′-ddC (AZddC), 3′-azido-2′,3′-ddG (AZddG), AZT, and 3′-azido-2′,3′-ddU (AZddU). The results indicate that mutations correlated with resistance to AZT (D67N/K70R/T215F/K219Q) confer resistance to the 3′-azidopyrimidine nucleosides (AZddC, AZT, and AZddU) but not to the 3′-azidopurine nucleosides (AZddA and AZddG). The data suggest that the presence of a 3′-azido group on the 3′-terminal nucleotide of the primer does not confer increased phosphorolytic excision by AZTR RT for all 3′-azido-ddNMP analogs. Thus, the 3′-azido group cannot be the only structural determinant important for the enhanced phosphorolytic excision of AZTMP associated with HIV resistance to AZT. Other structural components, such as the base, must play a role in defining the specificity of the excision phenotype arising from AZT resistance mutations.

Alkylglycerol Prodrugs of Phosphonoformate Are Potent In Vitro Inhibitors of Nucleoside-Resistant Human Immunodeficiency Virus Type 1 and Select for Resistance Mutations That Suppress Zidovudine Resistance

ABSTRACT Phosphonoformate (foscarnet; PFA) is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), but its use for the treatment of HIV-1 infection is limited by toxicity and the lack of an orally bioavailable formulation. Alkylglycerol-conjugated prodrugs of PFA (1-O-octadecyl-sn-glycero-3-PFA [B-PFA]) having sn-2 substituents of hydrogen (deoxybatyl-PFA [DB-PFA]), methyl (MB-PFA), or ethyl (EB-PFA) are more-potent inhibitors of wild-type HIV-1 in vitro than unmodified PFA and are orally bioavailable in mice. We have evaluated the activities of these compounds against a panel of nucleoside-resistant HIV-1 variants and have characterized the resistant variants that emerge following in vitro selection with the prodrugs. Except for an HIV-1 variant encoding the K65R mutation in RT that exhibited 3.3- to 8.2-fold resistance, the nucleoside-resistant viruses included in the panel were sensitive to the PFA prodrugs (<3-fold increase in 50% inhibitory concentration), including multinucleoside-resistant variants encoding the Q151M complex of mutations or the T69S[SA] insert. Viruses resistant to the PFA prodrugs (>10-fold) were selected in vitro after 15 or more serial passages of HIV-1 in MT-2 cells in escalating prodrug concentrations. Mutations detected in the resistant viruses were S117T, F160Y, and L214F (DB-PFA); M164I and L214F (MB-PFA); and W88G and L214F (EB-PFA). The S117T, F160Y, and M164I mutations have not been previously identified. Generation of recombinant viruses encoding the single and double mutations confirmed their roles in prodrug resistance, including 214F, which generally increased the level of resistance. When introduced into a zidovudine (AZT)-resistant background (67N 70R 215F 219Q), the W88G, S117T, F160Y, and M164I mutations reversed AZT resistance. This suppression of AZT resistance is consistent with the effects of other foscarnet resistance mutations that reduce ATP-dependent removal of AZT monophosphate from terminated template primers. The favorable activity and resistance profiles of these PFA prodrugs warrant their further evaluation as clinical candidates.

Improved Single Copy Assays for Quantification of Persistent HIV-1 Viremia in Patients on Suppressive Antiretroviral Therapy

ABSTRACT A quantitative real-time PCR (qRT-PCR) assay with single-copy sensitivity targeting HIV-1 gag RNA (the gag single-copy assay [gSCA]) has been used widely to quantify plasma viremia below the limit of detection of clinical assays in patients on effective antiretroviral therapy (ART), but viral RNA in 15 to 30% of samples amplifies inefficiently because of primer/probe mismatches. We sought to develop improved single-copy assays with increased sensitivity by improving nucleic acid recovery, designing qRT-PCR primers and a probe for a highly conserved region of integrase in the HIV-1 pol gene (the integrase single-copy assay [iSCA]), and increasing the plasma volume tested (Mega-iSCA). We evaluated gSCA versus iSCA in paired plasma samples from 10 consecutive patients with viremia of >1,000 copies/ml and 25 consecutive patients on suppressive ART. Three of 10 viremic samples amplified inefficiently with gSCA compared to the Roche Cobas Ampliprep/TaqMan 2.0, whereas all 10 samples amplified efficiently with iSCA. Among 25 samples from patients on suppressive ART, 8 of 12 samples that were negative for HIV-1 RNA by gSCA had detectable HIV-1 RNA by iSCA, and iSCA detected 3-fold or higher HIV-1 RNA levels compared to gSCA in 10 of 25 samples. Large-volume plasma samples (>20 ml) from 7 patients were assayed using Mega-iSCA, and HIV-1 RNA was quantifiable in 6, including 4 of 5 that were negative by standard-volume iSCA. These improved assays with superior sensitivity will be useful for evaluating whether in vivo interventions can reduce plasma viremia and for assessing relationships between residual viremia and other virologic parameters, including the inducible proviral reservoir.

Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA

ABSTRACT Although a number of PCR-based quantitative assays for measuring HIV-1 persistence during suppressive antiretroviral therapy (ART) have been reported, a simple, sensitive, reproducible method is needed for application to large clinical trials. We developed novel quantitative PCR assays for cell-associated (CA) HIV-1 DNA and RNA, targeting a highly conserved region in HIV-1 pol, with sensitivities of 3 to 5 copies/1 million cells. We evaluated the performance characteristics of the assays using peripheral blood mononuclear cells (PBMCs) from 5 viremic patients and 20 patients receiving effective ART. Total and resting CD4+ T cells were isolated from a subset of patients and tested for comparison with PBMCs. The estimated standard deviations including interassay variability and intra-assay variability of the assays were modest, i.e., 0.15 and 0.10 log10 copies/106 PBMCs, respectively, for CA HIV-1 DNA and 0.40 and 0.19 log10 copies/106 PBMCs for CA HIV-1 RNA. Testing of longitudinally obtained PBMC samples showed little variation for either viremic patients (median fold differences of 0.80 and 0.88 for CA HIV-1 DNA and RNA, respectively) or virologically suppressed patients (median fold differences of 1.14 and 0.97, respectively). CA HIV-1 DNA and RNA levels were strongly correlated (r = 0.77 to 1; P = 0.0001 to 0.037) for assays performed using PBMCs from different sources (phlebotomy versus leukapheresis) or using total or resting CD4+ T cells purified by either bead selection or flow cytometric sorting. Their sensitivity, reproducibility, and broad applicability to small numbers of mononuclear cells make these assays useful for observational and interventional studies that examine longitudinal changes in the numbers of HIV-1-infected cells and their levels of transcription.

In Vitro Selection and Analysis of Human Immunodeficiency Virus Type 1 Resistant to Derivatives of β-2′,3′-Didehydro-2′,3′-Dideoxy-5-Fluorocytidine

ABSTRACT Serial passage of human immunodeficiency virus type 1 in MT-2 cells in increasing concentrations of the d- and l-enantiomers of β-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (d4FC) resulted in the selection of viral variants with reverse transcriptase substitutions M184I or M184V for l-d4FC and I63L, K65R, K70N, K70E, or R172K for d-d4FC. Phenotypic analysis of site-directed mutants defined the role of these mutations in reducing susceptibility to l- or d-d4FC.

Anti-human immunodeficiency virus activity, cross-resistance, cytotoxicity, and intracellular pharmacology of the 3'-azido-2',3'-dideoxypurine nucleosides.

ABSTRACT 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.

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.