PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (39)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
more »
Document Types
1.  Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors 
A W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results.
doi:10.1128/AAC.02729-14
PMCID: PMC4136044  PMID: 24867966
2.  The R263K mutation in HIV integrase that is selected by dolutegravir may actually prevent clinically relevant resistance to this compound 
Journal of the International AIDS Society  2014;17(4Suppl 3):19518.
Introduction
Drug resistance against dolutegravir (DTG) or the nucleosides with which it has been co-administered has never been observed in patients receiving this drug in first-line therapy. In contrast, a R263K mutation that confers low-level resistance (3–4 fold) to DTG has been selected by DTG in culture. Our group has ascribed the absence of resistance to DTG to the high fitness cost exacted by the R263K mutation and an inability of HIV to generate compensatory mutations.
Materials and Methods
We generated recombinant integrase enzymes and viruses containing various combinations of mutations and studied these enzymatically and in culture. We also selected for resistance against raltegravir (RAL) using viruses containing the R263K mutation.
Results
The R263K mutation alone conferred an approximate 3-fold level of resistance to DTG and a 40% loss in viral replicative capacity and recombinant integrase activity. Secondary mutations selected at positions H51Y or E138K did not individually affect either enzyme activity or DTG resistance, but the combination of R263K together with H51Y or E138K increased DTG resistance to about 7-fold accompanied by a ≈75% loss in each of viral replication capacity, and both in vitro and in vivo integrase activity. Conversely, combinations of R263K together with multiple resistance mutations for RAL and/or EVG at positions 92,143, 148 and 155 resulted in even further diminished enzymatic activity that may be incompatible with viral survival. Modelling of the 3-dimensional structure of integrase suggests that R263K is located in a region that may not permit further mutagenesis if secondary mutations at H51Y or E138K are also present. Moreover, integrase that contains R263K together with substitutions at positions 92, 143, 148 and 155 may be enzymatically inactive. The use of the R263K-containing virus to select for resistance to RAL led to the appearance of RAL-containing mutations but the loss of R263K.
Conclusions
Secondary mutations to R263K following selection with DTG have all led to diminished viral and enzymatic fitness, helping to explain why resistance to DTG in previously drug-naïve subjects has never been observed. The use of DTG in first-line therapy may prevent the facile development of drug resistance and help to forestall ongoing HIV transmission.
doi:10.7448/IAS.17.4.19518
PMCID: PMC4224880  PMID: 25394027
3.  HIV-1 group O integrase displays lower susceptibility to raltegravir and has a different mutational pathway for resistance than HIV-1 group M 
Journal of the International AIDS Society  2014;17(4Suppl 3):19738.
Introduction
HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations.
Viruses and Methods
We cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A) and B (HIV-O/B), a HIV-O divergent strain (HIV-O/Div), and HIV-1 group M (subtype B, HIV-M/B) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki) and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs.
Results
HIV-O integrase and viruses were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1) mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL), T66I, E92Q, E157Q (EVG) and M50I, R263K (DTG) and (2) signature mutations for HIV-O (i.e. not described in HIV-M) F121C (HIV-O/B for RAL), V75I (HIV-O/A for RAL) and S153V (HIV-O/A for DTG). Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to valine (153AGGC→153VGTC) is easier than substitution of alanine to tyrosine (153AGGC→153YTAC), with only a transversion needed instead of one transition plus one transversion.
Conclusions
This is the first report of susceptibility and resistance in vitro to INSTIs for HIV-O. Our study confirmed the impact of HIV-O polymorphism, on susceptibility to INSTIs and the emergence of resistance mutations.
doi:10.7448/IAS.17.4.19738
PMCID: PMC4225329  PMID: 25397483
4.  The Connection Domain Mutation N348I in HIV-1 Reverse Transcriptase Enhances Resistance to Etravirine and Rilpivirine but Restricts the Emergence of the E138K Resistance Mutation by Diminishing Viral Replication Capacity 
Journal of Virology  2014;88(3):1536-1547.
Clinical resistance to rilpivirine (RPV), a novel nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI), is associated an E-to-K mutation at position 138 (E138K) in RT together with an M184I/V mutation that confers resistance against emtricitabine (FTC), a nucleoside RT inhibitor (NRTI) that is given together with RPV in therapy. These two mutations can compensate for each other in regard to fitness deficits conferred by each mutation alone, raising the question of why E138K did not arise spontaneously in the clinic following lamivudine (3TC) use, which also selects for the M184I/V mutations. In this context, we have investigated the role of a N348I connection domain mutation that is prevalent in treatment-experienced patients. N348I confers resistance to both the NRTI zidovudine (ZDV) and the NNRTI nevirapine (NVP) and was also found to be associated with M184V and to compensate for deficits associated with the latter mutation. Now, we show that both N348I alone and N348I/M184V can prevent or delay the emergence of E138K under pressure with RPV or a related NNRTI, termed etravirine (ETR). N348I also enhanced levels of resistance conferred by E138K against RPV and ETR by 2.2- and 2.3-fold, respectively. The presence of the N348I or M184V/N348I mutation decreased the replication capacity of E138K virus, and biochemical assays confirmed that N348I, in a background of E138K, impaired RT catalytic efficiency and RNase H activity. These findings help to explain the low viral replication capacity of viruses containing the E138K/N348I mutations and how N348I delayed or prevented the emergence of E138K in patients with M184V-containing viruses.
doi:10.1128/JVI.02904-13
PMCID: PMC3911599  PMID: 24227862
5.  Role of the K101E Substitution in HIV-1 Reverse Transcriptase in Resistance to Rilpivirine and Other Nonnucleoside Reverse Transcriptase Inhibitors 
Antimicrobial Agents and Chemotherapy  2013;57(11):5649-5657.
Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.
doi:10.1128/AAC.01536-13
PMCID: PMC3811317  PMID: 24002090
6.  Basis for Early and Preferential Selection of the E138K Mutation in HIV-1 Reverse Transcriptase 
Antimicrobial Agents and Chemotherapy  2013;57(10):4681-4688.
E138K, a G→A mutation in HIV-1 reverse transcriptase (RT), is preferentially selected by etravirine (ETR) and rilpivirine over other substitutions at position E138 that offer greater drug resistance. We hypothesized that there was a mutational bias for the E138K substitution and designed an allele-specific PCR to monitor the emergence of E138A/G/K/Q/R/V during ETR selection experiments. We also performed competition experiments using mutated viruses and quantified the prevalence of E138 minority species in drug-naive patients. E138K, as well as E138G, consistently emerged first during ETR selection experiments, followed by E138A and E138Q; E138R was never selected. Surprisingly, E138K was identified as a tiny minority in 23% of drug-naive subtype B patients, a result confirmed by ultradeep sequencing (UDS). This result could reflect a low fitness cost of E138K; however, E138K was one of the least fit substitutions at codon E138, even after taking into account the deoxynucleoside triphosphate pools of the cells used in competition experiments. Further UDS analysis revealed other minority species in a pattern consistent with the mutational bias of HIV RT. There was no evidence of APOBEC3-hypermutation in these selection experiments or in patients. Our results confirm the mutational bias of HIV-1 in patients and highlight the importance of G→A mutations in HIV-1 drug resistance evolution.
doi:10.1128/AAC.01029-13
PMCID: PMC3811420  PMID: 23856772
7.  Effect of Mutations at Position E138 in HIV-1 Reverse Transcriptase and Their Interactions with the M184I Mutation on Defining Patterns of Resistance to Nonnucleoside Reverse Transcriptase Inhibitors Rilpivirine and Etravirine 
Impacts of mutations at position E138 (A/G/K/Q/R/V) alone or in combination with M184I in HIV-1 reverse transcriptase (RT) were investigated. We also determined why E138K is the most prevalent nonnucleoside reverse transcriptase inhibitor mutation in patients failing rilpivirine (RPV) therapy. Recombinant RT enzymes and viruses containing each of the above-mentioned mutations were generated, and drug susceptibility was assayed. Each of the E138A/G/K/Q/R mutations, alone or in combination with M184I, resulted in decreased susceptibility to RPV and etravirine (ETR). The maximum decrease in susceptibility to RPV was observed for E138/R/Q/G by both recombinant RT assay and cell-based assays. E138Q/R-containing enzymes and viruses also showed the most marked decrease in susceptibility to ETR by both assays. The addition of M184I to the E138 mutations did not significantly change the levels of diminution in drug susceptibility. These findings indicate that E138R caused the highest level of loss of susceptibility to both RPV and ETR, and, accordingly, E138R should be recognized as an ETR resistance-associated mutation. The E138K/Q/R mutations can compensate for M184I in regard to both enzymatic fitness and viral replication capacity. The favored emergence of E138K over other mutations at position E138, together with M184I, is not due to an advantage in either the level of drug resistance or viral replication capacity but may reflect the fact that E138R and E138Q require two distinct mutations to occur, one of which is a disfavorable G-to-C mutation, whereas E138K requires only a single favorable G-to-A hypermutation. Of course, other factors may also affect the concept of barrier to resistance.
doi:10.1128/AAC.00348-13
PMCID: PMC3697388  PMID: 23612196
8.  Molecular Mechanism of Antagonism between the Y181C and E138K Mutations in HIV-1 Reverse Transcriptase 
Journal of Virology  2012;86(23):12983-12990.
Etravirine (ETR) is an expanded-spectrum nonnucleoside reverse transcriptase inhibitor (NNRTI) approved for use as an antiretroviral agent in treatment-experienced patients. Y181C and E138K in HIV-1 RT are among 20 different drug resistance mutations associated with ETR. However, E138K can be consistently selected by ETR when wild-type viruses but not viruses containing Y181C are grown in tissue culture. This study was carried out to evaluate any possible mechanisms that might explain antagonism between the Y181C and E138K mutations. Accordingly, we performed tissue culture studies to investigate the evolutionary dynamics of E138K in both a wild-type (WT) and a Y181C background. We also generated recombinant enzymes containing Y181C and E138K alone or in combination in order to study enzyme processivity, rates of processive DNA synthesis, enzyme kinetics, and susceptibility to ETR. We now show that the presence of the Y181C mutation prevented the emergence of E138K in cell culture and that the simultaneous presence of E138K and Y181C impaired each of enzyme activity, processivity, rate of processive DNA synthesis, and deoxynucleoside triphosphate (dNTP) affinity. The addition of E138K to Y181C also decreased the level of resistance to ETR compared to that obtained with Y181C alone.
doi:10.1128/JVI.02005-12
PMCID: PMC3497622  PMID: 22993165
9.  Maraviroc and Other HIV-1 Entry Inhibitors Exhibit a Class-Specific Redistribution Effect That Results in Increased Extracellular Viral Load 
HIV entry inhibitors, such as maraviroc (MVC), prevent cell-free viruses from entering the cells. In clinical trials, patients who were treated with MVC often displayed viral loads that were above the limit of conventional viral load detection compared to efavirenz-based regimens. We hypothesize that viruses blocked by entry inhibitors may be redistributed to plasma, where they artificially increase viral load measurements compared to those with the use of antiretroviral drugs (ARVs) that act intracellularly. We infected PM-1 cells with CCR5-tropic HIV-1 BaL or CXCR4-tropic HIV-1 NL4-3 in the presence of inhibitory concentrations of efavirenz, raltegravir, enfuvirtide, maraviroc, and AMD3100, the latter three being entry inhibitors. Supernatant viral load, reverse transcriptase enzyme activity, and intracellular nucleic acid levels were measured at times up to 24 h postinfection. Infectivity of redistributed dual-tropic HIV-1 was assessed using TZM-bl cells. Extracellular viral load analysis revealed that entry inhibitor-treated cells had higher levels of virus in the supernatant than the cells treated with other ARVs at 8 h postinfection. By 24 h, the supernatant viral load was still higher for entry inhibitors than other ARVs. We observed a correlation between viral load and the step of entry inhibition. Dual-tropic virus infectivity was undiminished utilizing the CCR5 coreceptor following redistribution by CXCR4 entry inhibition. This in vitro model indicates that entry inhibitors exhibit a redistribution effect unseen with intracellular ARV drugs. Based on these results, the effectiveness of some entry inhibitors may be underestimated if plasma viral load is used as a sole indicator of clinical success.
doi:10.1128/AAC.00409-12
PMCID: PMC3421589  PMID: 22615275
10.  HIV gp120 H375 Is Unique to HIV-1 Subtype CRF01_AE and Confers Strong Resistance to the Entry Inhibitor BMS-599793, a Candidate Microbicide Drug 
BMS-599793 is a small molecule entry inhibitor that binds to human immunodeficiency virus type 1 (HIV-1) gp120, resulting in the inhibition of CD4-dependent entry into cells. Since BMS-599793 is currently considered a candidate microbicide drug, we evaluated its efficacy against a number of primary patient HIV isolates from different subtypes and circulating recombinant forms (CRFs) and showed that activity varied between ∼3 ρM and 7 μM at 50% effective concentrations (EC50s). Interestingly, CRF01_AE HIV-1 isolates consistently demonstrated natural resistance against this compound. Genotypic analysis of >1,600 sequences (Los Alamos HIV sequence database) indicated that a single amino acid polymorphism in Env, H375, may account for the observed BMS-599793 resistance in CRF01_AE HIV-1. Results of site-directed mutagenesis experiments confirmed this hypothesis, and in silico drug docking simulations identified a drug resistance mechanism at the molecular level. In addition, CRF01_AE viruses were shown to be resistant to multiple broadly neutralizing monoclonal antibodies. Thus, our results not only provide insight into how Env polymorphisms may contribute to entry inhibitor resistance but also may help to elucidate how HIV can evade some broadly neutralizing antibodies. Furthermore, the high frequency of H375 in CRF01_AE HIV-1, and its apparent nonoccurrence in other subtypes, could serve as a means for rapid identification of CRF01_AE infections.
doi:10.1128/AAC.00639-12
PMCID: PMC3421599  PMID: 22615295
11.  Subunit-Selective Mutational Analysis and Tissue Culture Evaluations of the Interactions of the E138K and M184I Mutations in HIV-1 Reverse Transcriptase 
Journal of Virology  2012;86(16):8422-8431.
The emergence of HIV-1 drug resistance remains a major obstacle in antiviral therapy. M184I/V and E138K are signature mutations of clinical relevance in HIV-1 reverse transcriptase (RT) for the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and emtricitabine (FTC) and the second-generation (new) nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV), respectively, and the E138K mutation has also been shown to be selected by etravirine in cell culture. The E138K mutation was recently shown to compensate for the low enzyme processivity and viral fitness associated with the M184I/V mutations through enhanced deoxynucleoside triphosphate (dNTP) usage, while the M184I/V mutations compensated for defects in polymerization rates associated with the E138K mutations under conditions of high dNTP concentrations. The M184I mutation was also shown to enhance resistance to RPV and ETR when present together with the E138K mutation. These mutual compensatory effects might also enhance transmission rates of viruses containing these two mutations. Therefore, we performed tissue culture studies to investigate the evolutionary dynamics of these viruses. Through experiments in which E138K-containing viruses were selected with 3TC-FTC and in which M184I/V viruses were selected with ETR, we demonstrated that ETR was able to select for the E138K mutation in viruses containing the M184I/V mutations and that the M184I/V mutations consistently emerged when E138K viruses were selected with 3TC-FTC. We also performed biochemical subunit-selective mutational analyses to investigate the impact of the E138K mutation on RT function and interactions with the M184I mutation. We now show that the E138K mutation decreased rates of polymerization, impaired RNase H activity, and conferred ETR resistance through the p51 subunit of RT, while an enhancement of dNTP usage as a result of the simultaneous presence of both mutations E138K and M184I occurred via both subunits.
doi:10.1128/JVI.00271-12
PMCID: PMC3421741  PMID: 22623801
12.  Characterization of the R263K Mutation in HIV-1 Integrase That Confers Low-Level Resistance to the Second-Generation Integrase Strand Transfer Inhibitor Dolutegravir 
Journal of Virology  2012;86(5):2696-2705.
Integrase (IN) strand transfer inhibitors (INSTIs) have been developed to inhibit the ability of HIV-1 integrase to irreversibly link the reverse-transcribed viral DNA to the host genome. INSTIs have proven their high efficiency in inhibiting viral replication in vitro and in patients. However, first-generation INSTIs have only a modest genetic barrier to resistance, allowing the virus to escape these powerful drugs through several resistance pathways. Second-generation INSTIs, such as dolutegravir (DTG, S/GSK1349572), have been reported to have a higher resistance barrier, and no novel drug resistance mutation has yet been described for this drug. Therefore, we performed in vitro selection experiments with DTG using viruses of subtypes B, C, and A/G and showed that the most common mutation to emerge was R263K. Further analysis by site-directed mutagenesis showed that R263K does confer low-level resistance to DTG and decreased integration in cell culture without altering reverse transcription. Biochemical cell-free assays performed with purified IN enzyme containing R263K confirmed the absence of major resistance against DTG and showed a slight decrease in 3′ processing and strand transfer activities compared to the wild type. Structural modeling suggested and in vitro IN-DNA binding assays show that the R263K mutation affects IN-DNA interactions.
doi:10.1128/JVI.06591-11
PMCID: PMC3302270  PMID: 22205735
13.  In Vitro Resistance Profile of the Candidate HIV-1 Microbicide Drug Dapivirine 
Antiretroviral-based microbicides may offer a means to reduce the sexual transmission of HIV-1. Suboptimal use of a microbicide may, however, lead to the development of drug resistance in users that are already, or become, infected with HIV-1. In such cases, the efficacy of treatments may be compromised since the same (or similar) antiretrovirals used in treatments are being developed as microbicides. To help predict which drug resistance mutations may develop in the context of suboptimal use, HIV-1 primary isolates of different subtypes and different baseline resistance profiles were used to infect primary cells in vitro in the presence of increasing suboptimal concentrations of the two candidate microbicide antiretrovirals dapivirine (DAP) and tenofovir (TFV) alone or in combination. Infections were ongoing for 25 weeks, after which reverse transcriptase genotypes were determined and scrutinized for the presence of any clinically recognized reverse transcriptase drug resistance mutations. Results indicated that suboptimal concentrations of DAP alone facilitated the emergence of common nonnucleoside reverse transcriptase inhibitor resistance mutations, while suboptimal concentrations of DAP plus TFV gave rise to fewer mutations. Suboptimal concentrations of TFV alone did not frequently result in the development of resistance mutations. Sensitivity evaluations for stavudine (d4T), nevirapine (NVP), and lamivudine (3TC) revealed that the selection of resistance as a consequence of suboptimal concentrations of DAP may compromise the potential for NVP to be used in treatment, a finding of potential relevance in developing countries.
doi:10.1128/AAC.05821-11
PMCID: PMC3264246  PMID: 22123692
14.  Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase for Deficits in Viral Replication Capacity and Enzyme Processivity Associated with the M184I/V Mutations▿  
Journal of Virology  2011;85(21):11300-11308.
Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two nucleos(t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse transcriptase (RT) enzymes and HIV-1NL4-3 infectious clones. Drug susceptibilities were determined in cord blood mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV), and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state kinetic analysis demonstrated that the E138K mutation resulted in decreased Kms for dNTPs. In contrast, M184I/V resulted in an increased Km for dNTPs compared to those for WT RT. These results indicate that the E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.
doi:10.1128/JVI.05584-11
PMCID: PMC3194954  PMID: 21849444
15.  Characterization of the E138K Resistance Mutation in HIV-1 Reverse Transcriptase Conferring Susceptibility to Etravirine in B and Non-B HIV-1 Subtypes ▿  
We have selected for resistance to etravirine (ETR) and efavirenz (EFV) in tissue culture using three subtype B, three subtype C, and two CRF02_AG clinical isolates, grown in cord blood mononuclear cells. Genotypic analysis was performed at baseline and at various weeks of selection. Phenotypic resistance in regard to ETR, EFV, and nevirapine (NVP) was evaluated at weeks 25 to 30 for all ETR-selected viruses and in viral clones that contained specific resistance mutations that were inserted by site-directed mutagenesis into pNL-4.3 and AG plasmids. The results show that ETR selected mutations at positions V90I, K101Q, E138K, V179D/E/F, Y181C, V189I, G190E, H221H/Y, and M230L and that E138K was the first of these to emerge in most instances. The time to the emergence of resistance was longer in the case of ETR (18 weeks) compared to EFV (11 weeks), and no differences in the patterns of emergent mutations could be documented between the B and non-B subtypes. Viral clones containing E138K displayed low-level phenotypic resistance to ETR (3.8-fold) and modestly impaired replication capacity (2-fold) compared to wild-type virus. ETR-selected virus showed a high degree of cross-resistance to NVP but not to EFV. We identified K101Q, E138K, V179E, V189I, G190E, and H221Y as mutations not included among the 17 currently recognized resistance-associated mutations for ETR.
doi:10.1128/AAC.01192-10
PMCID: PMC3028807  PMID: 21135184
16.  A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1 
PLoS ONE  2011;6(5):e20208.
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.
doi:10.1371/journal.pone.0020208
PMCID: PMC3105016  PMID: 21655292
17.  Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor ▿  
Journal of Virology  2010;84(18):9210-9216.
MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg2+ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.
doi:10.1128/JVI.01164-10
PMCID: PMC2937597  PMID: 20610719
18.  HIV-1 Protease Codon 36 Polymorphisms and Differential Development of Resistance to Nelfinavir, Lopinavir, and Atazanavir in Different HIV-1 Subtypes▿  
The amino acid at position 36 of the HIV-1 protease differs among various viral subtypes, in that methionine is usually found in subtype B viruses but isoleucine is common in other subtypes. This polymorphism is associated with higher rates of treatment failure involving protease inhibitors (PIs) in non-subtype B-infected patients. To investigate this, we generated genetically homogeneous wild-type viruses from subtype B, subtype C, and CRF02_AG full-length molecular clones and showed that subtype C and CRF02_AG I36 viruses exhibited higher levels of resistance to various PIs than their respective M36 counterparts, while the opposite was observed for subtype B viruses. Selections for resistance with each variant were performed with nelfinavir (NFV), lopinavir (LPV), and atazanavir (ATV). Sequence analysis of the protease gene at week 35 revealed that the major NFV resistance mutation D30N emerged in NFV-selected subtype B viruses and in I36 subtype C viruses, despite polymorphic variation. A unique mutational pattern developed in subtype C M36 viruses selected with NFV or ATV. The presence of I47A in LPV-selected I36 CRF02_AG virus conferred higher-level resistance than L76V in LPV-selected M36 CRF02_AG virus. Phenotypic analysis revealed a >1,000-fold increase in NFV resistance in I36 subtype C NFV-selected virus with no apparent impact on viral replication capacity. Thus, the position 36 polymorphism in the HIV-1 protease appears to have a differential effect on both drug susceptibility and the viral replication capacity, depending on both the viral subtype and the drug being evaluated.
doi:10.1128/AAC.01828-09
PMCID: PMC2897293  PMID: 20404123
19.  The M230L Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation in HIV-1 Reverse Transcriptase Impairs Enzymatic Function and Viral Replicative Capacity▿  
The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.
doi:10.1128/AAC.01795-09
PMCID: PMC2876396  PMID: 20308384
20.  Comparative biochemical analysis of recombinant reverse transcriptase enzymes of HIV-1 subtype B and subtype C 
Retrovirology  2010;7:80.
Background
HIV-1 subtype C infections account for over half of global HIV infections, yet the vast focus of HIV-1 research has been on subtype B viruses which represent less than 12% of the global pandemic. Since HIV-1 reverse transcriptase (RT) is a major target of antiviral therapy, and since differential drug resistance pathways have been observed among different HIV subtypes, it is important to study and compare the enzymatic activities of HIV-1 RT derived from each of subtypes B and C as well as to determine the susceptibilities of these enzymes to various RT inhibitors in biochemical assays.
Methods
Recombinant subtype B and C HIV-1 RTs in heterodimeric form were purified from Escherichia coli and enzyme activities were compared in cell-free assays. The efficiency of (-) ssDNA synthesis was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Processivity was assayed under single-cycle conditions using both homopolymeric and heteropolymeric RNA templates. Intrinsic RNase H activity was compared using 5'-end labeled RNA template annealed to 3'-end recessed DNA primer in a time course study in the presence and absence of a heparin trap. A mis-incorporation assay was used to assess the fidelity of the two RT enzymes. Drug susceptibility assays were performed both in cell-free assays using recombinant enzymes and in cell culture phenotyping assays.
Results
The comparative biochemical analyses of recombinant subtype B and subtype C HIV-1 reverse transcriptase indicate that the two enzymes are very similar biochemically in efficiency of tRNA-primed (-) ssDNA synthesis, processivity, fidelity and RNase H activity, and that both enzymes show similar susceptibilities to commonly used NRTIs and NNRTIs. Cell culture phenotyping assays confirmed these results.
Conclusions
Overall enzyme activity and drug susceptibility of HIV-1 subtype C RT are comparable to those of subtype B RT. The use of RT inhibitors (RTIs) against these two HIV-1 enzymes should have comparable effects.
doi:10.1186/1742-4690-7-80
PMCID: PMC2959035  PMID: 20929562
21.  Human Immunodeficiency Virus Type 1 Recombinant Reverse Transcriptase Enzymes Containing the G190A and Y181C Resistance Mutations Remain Sensitive to Etravirine▿  
Antimicrobial Agents and Chemotherapy  2009;53(11):4667-4672.
Etravirine (ETR) is a second-generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) active against common human immunodeficiency virus type 1 (HIV-1) drug-resistant strains. This study was designed to determine the extent to which each of the Y181C or G190A mutations in RT might confer resistance to ETR and other members of the NNRTI family of drugs. Recombinant HIV-1 RT enzymes containing either the Y181C or the G190A mutation, or both mutations in tandem, were purified. Both RNA- and DNA-dependent DNA polymerase assays were performed in order to determine the extent to which each of these mutations might confer resistance in cell-free biochemical assays against each of ETR, efavirenz, and nevirapine. Both the biochemical and the cell-based phenotypic assays confirmed the susceptibility of G190A-containing enzymes and viruses to ETR. The results of this study indicate that the G190A mutation is not associated with resistance to ETR.
doi:10.1128/AAC.00800-09
PMCID: PMC2772356  PMID: 19704127
22.  Transmission Networks of Drug Resistance Acquired in Primary/Early Stage HIV Infection 
AIDS (London, England)  2008;22(18):2509-2515.
OBJECTIVES
Population-based sequencing of primary/recent HIV infections (PHI) can provide a framework for understanding transmission dynamics of local epidemics. In Quebec, half of PHI represent clustered transmission events. This study ascertained the cumulative implications of clustering on onward transmission of drug resistance.
METHODS
HIV-1 pol sequence datasets were available for all genotyped PHI (<6 months post-seroconversion (n=848 subtype B infections, 1997-2007). Phylogenetic analysis established clustered transmission events, based on maximum likelihood topologies having high bootstrap values (>98%) and short genetic distances. The distributions of resistance to nucleoside and non-nucleoside RT inhibitors (NRTIs and NNRTIs) and protease inhibitors (PIs) in unique and clustered transmissions were ascertained.
Results
Episodic clustering was observed in half of recent/early stage infections from 1997-2008. Overall, 29% and 28% of new infections segregated into small (<5 PHI/cluster, n=242/848) and large transmission chains (≥5 PHI/cluster, n=239/848), averaging 2.8 ± 0.1 PHI/cluster and 10.3 ± 1.0 PHI/cluster, respectively. The transmission of nucleoside analogue mutations and 215 resistant variants (T215C/D/I/F/N/S/Y) declined with clustering (7.9% vs. 3.4% vs. 1.2% and 5.8% vs. 1.7% vs. 1.1% for unique, small and large clustered transmissions, respectively). In contrast, clustering was associated with the increased transmission of viruses harbouring resistance to NNRTIs (6.6% vs. 6.0% vs. 15.5%, respectively).
CONCLUSIONS
Clustering in early/PHI stage infection differentially affects transmission of drug resistance to different drug classes. Public health, prevention and diagnostic strategies, targeting PHI, afford a unique opportunity to curb the spread of transmitted drug resistance.
doi:10.1097/QAD.0b013e3283121c90
PMCID: PMC2650396  PMID: 19005274
23.  Comparative biochemical analysis of HIV-1 subtype B and C integrase enzymes 
Retrovirology  2009;6:103.
Background
Integrase inhibitors are currently being incorporated into highly active antiretroviral therapy (HAART). Due to high HIV variability, integrase inhibitor efficacy must be evaluated against a range of integrase enzymes from different subtypes.
Methods
This study compares the enzymatic activities of HIV-1 integrase from subtypes B and C as well as susceptibility to various integrase inhibitors in vitro. The catalytic activities of both enzymes were analyzed in regard to each of 3' processing and strand transfer activities both in the presence and absence of the integrase inhibitors raltegravir (RAL), elvitegravir (EVG), and MK-2048.
Results
Our results show that integrase function is similar with enzymes of either subtype and that the various integrase strand transfer inhibitors (INSTIs) that were employed possessed similar inhibitory activity against both enzymes.
Conclusion
This suggests that the use of integrase inhibitors against HIV-1 subtype C will result in comparable outcomes to those obtained against subtype B infections.
doi:10.1186/1742-4690-6-103
PMCID: PMC2779801  PMID: 19906306
24.  Apricitabine Does Not Select Additional Drug Resistance Mutations in Tissue Culture in Human Immunodeficiency Virus Type 1 Variants Containing K65R, M184V, or M184V plus Thymidine Analogue Mutations▿  
Human immunodeficiency virus type 1 containing the reverse transcriptase mutation M184V or K65R or mutations M41L, M184V, and T215Y did not accumulate additional resistance mutations in the reverse transcriptase when increasing amounts of apricitabine drug pressure were applied. The original mutations were maintained by the presence of apricitabine but were lost when cultured without drug pressure.
doi:10.1128/AAC.01168-08
PMCID: PMC2663123  PMID: 19223637
25.  Detection of Human Immunodeficiency Virus (HIV) Type 1 M184V and K103N Minority Variants in Patients with Primary HIV Infection▿  
We used an allele-specific real-time PCR assay to explore the presence of K103N and M184V minority species among primary human immunodeficiency virus (HIV) infections and their potential influence in HIV transmission. Thirty randomly chosen antiretroviral drug-naive patients lacking both the K103N and the M184V mutations as determined by conventional sequencing methods were studied, and K103N and M184V viral minority species were found in three (10%) and four (11%) patients, respectively.
doi:10.1128/AAC.01494-08
PMCID: PMC2663097  PMID: 19171798

Results 1-25 (39)