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1.  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.
PMCID: PMC3811420  PMID: 23856772
2.  Latent HIV-1 Can Be Reactivated by Cellular Superinfection in a Tat-Dependent Manner, Which Can Lead to the Emergence of Multidrug-Resistant Recombinant Viruses 
Journal of Virology  2013;87(17):9620-9632.
The HIV-1 latent reservoir represents an important source of genetic diversity that could contribute to viral evolution and multidrug resistance following latent virus reactivation. This could occur by superinfection of a latently infected cell. We asked whether latent viruses might be reactivated when their host cells are superinfected, and if so, whether they could contribute to the generation of recombinant viruses. Using populations of latently infected Jurkat cells, we found that latent viruses were efficiently reactivated upon superinfection. Pathways leading to latent virus reactivation via superinfection might include gp120-CD4/CXCR4-induced signaling, modulation of the cellular environment by Nef, and/or the activity of Tat produced upon superinfection. Using a range of antiviral compounds and genetic approaches, we show that gp120 and Nef are not required for latent virus reactivation by superinfection, but this process depends on production of functional Tat by the superinfecting virus. In a primary cell model of latency in unstimulated CD4 T cells, superinfection also led to latent virus reactivation. Drug-resistant latent viruses were also reactivated following superinfection in Jurkat cells and were able to undergo recombination with the superinfecting virus. Under drug-selective pressure, this generated multidrug-resistant recombinants that were identified by unique restriction digestion band patterns and by population-level sequencing. During conditions of poor drug adherence, treatment interruption or treatment failure, or in drug-impermeable sanctuary sites, reactivation of latent viruses by superinfection or other means could provide for the emergence or spread of replicatively fit viruses in the face of strong selective pressures.
PMCID: PMC3754111  PMID: 23804632
3.  Evaluation of a Cost Effective In-House Method for HIV-1 Drug Resistance Genotyping Using Plasma Samples 
PLoS ONE  2014;9(2):e87441.
Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.
The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity.
The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US) as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C) and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88) with known viral loads ranges from 1000–1.8 million RNA copies/ml.
Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$), thus making it cost effective.
The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.
PMCID: PMC3922725  PMID: 24533056
4.  Reviewer acknowledgement 2013 
Retrovirology  2014;11:5.
Contributing reviewers
Retrovirology would like to sincerely thank the following for giving their time and expertise to review manuscripts for the journal in 2013. Their support for the journal is greatly appreciated.
PMCID: PMC3916064
5.  Characterization of Antiviral Activity of Benzamide Derivative AH0109 against HIV-1 Infection 
In the absence of an effective vaccine against HIV-1 infection, anti-HIV-1 strategies play a major role in disease control. However, the rapid emergence of drug resistance against all currently used anti-HIV-1 molecules necessitates the development of new antiviral molecules and/or strategies against HIV-1 infection. In this study, we have identified a benzamide derivative named AH0109 that exhibits potent anti-HIV-1 activity at an 50% effective concentration of 0.7 μM in HIV-1-susceptible CD4+ C8166 T cells. Mechanistic analysis revealed that AH0109 significantly inhibits both HIV-1 reverse transcription and viral cDNA nuclear import. Furthermore, our infection experiments indicated that AH0109 is capable of disrupting the replication of HIV-1 strains that are resistant to the routinely used anti-HIV-1 drugs zidovudine, lamivudine, nevirapine, and raltegravir. Together, these findings provide evidence for a newly identified antiviral molecule that can potentially be developed as an anti-HIV-1 agent.
PMCID: PMC3719726  PMID: 23669388
6.  The M50I polymorphic substitution in association with the R263K mutation in HIV-1 subtype B integrase increases drug resistance but does not restore viral replicative fitness 
Retrovirology  2014;11:7.
First-generation integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG), have been clinically proven to be effective antiretrovirals for the treatment of HIV-positive patients. However, their relatively low genetic barrier for resistance makes them susceptible to the emergence of drug resistance mutations. In contrast, dolutegravir (DTG) is a newer INSTI that appears to have a high genetic barrier to resistance in vivo. However, the emergence of the resistance mutation R263K followed by the polymorphic substitution M50I has been observed in cell culture. The M50I polymorphism is also observed in 10-25% of INSTI-naïve patients and has been reported in combination with R263K in a patient failing treatment with RAL.
Using biochemical cell-free strand-transfer assays and resistance assays in TZM-bl cells, we demonstrate that the M50I polymorphism in combination with R263K increases resistance to DTG in tissue culture and in biochemical assays but does not restore the viral fitness cost associated with the R263K mutation.
Since the combination of the R263K mutation and the M50I polymorphism results in a virus with decreased viral fitness and limited cross-resistance, the R263K resistance pathway may represent an evolutionary dead-end. Although this hypothesis has not yet been proven, it may be more advantageous to treat HIV-positive individuals with DTG in first-line than in second or third-line therapy.
PMCID: PMC3898230  PMID: 24433497
HIV integrase; Subtype B; Antiretrovirals; R263K; Resistance mutation; M50I; Polymorphism; INSTI-naïve
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.
PMCID: PMC3697388  PMID: 23612196
8.  Productive Entry of HIV-1 during Cell-to-Cell Transmission via Dynamin-Dependent Endocytosis 
Journal of Virology  2013;87(14):8110-8123.
HIV-1 can be transmitted as cell-free virus or via cell-to-cell contacts. Cell-to-cell transmission between CD4+ T cells is the more efficient mode of transmission and is predominant in lymphoid tissue, where the majority of virus resides. Yet the cellular mechanisms underlying productive cell-to-cell transmission in uninfected target cells are unclear. Although it has been demonstrated that target cells can take up virus via endocytosis, definitive links between this process and productive infection remain undefined, and this route of transmission has been proposed to be nonproductive. Here, we report that productive cell-to-cell transmission can occur via endocytosis in a dynamin-dependent manner and is sensitive to clathrin-associated antagonists. These data were obtained in a number of CD4+ T-cell lines and in primary CD4+ T cells, using both CXCR4- and CCR5-tropic virus. However, we also found that HIV-1 demonstrated flexibility in its use of such endocytic pathways as certain allogeneic transmissions were seen to occur in a dynamin-dependent manner but were insensitive to clathrin-associated antagonists. Also, depleting cells of the clathrin accessory protein AP180 led to a viral uptake defect associated with enhanced infection. Collectively, these data demonstrate that endosomal uptake of HIV-1 during cell-to-cell transmission leads to productive infection, but they are also indicative of a flexible model of viral entry during cell-to-cell transmission, in which the virus can alter its entry route according to the pressures that it encounters.
PMCID: PMC3700189  PMID: 23678185
9.  What if HIV were unable to develop resistance against a new therapeutic agent? 
BMC Medicine  2013;11:249.
The HIV integrase inhibitor, Dolutegravir (DTG), was recently approved by the Food and Drug Administration in the United States and is the only HIV drug that has not selected for resistance mutations in the clinic when used as part of first-line therapy. This has led to speculation that DTG might have a higher genetic barrier for the development of drug resistance than the other compounds that are used in therapy.
In this Opinion article, we speculate that this is due to greatly diminished replication capacity on the part of viruses that might become resistant to DTG when the drug is used in initial therapy and that DTG might be able to be used in HIV prevention and eradication strategies. We also note that no compensatory mutation that might restore viral replication fitness to HIV in the aftermath of the appearance of a single drug resistance mutation has yet to be observed.
DTG is a valuable addition to the anti-HIV armamentarium of drugs and its long-term utility may potentially exceed its obvious use in treatment of HIV disease.
PMCID: PMC3842747  PMID: 24267867
Human immunodeficiency virus type 1; Integrase inhibitors; Antiretroviral therapy; Dolutegravir; HIV prevention strategies; Viral fitness; Drug resistance
10.  World AIDS Day: together we will stop HIV transmission and conquer AIDS 
Retrovirology  2013;10:129.
PMCID: PMC3828426  PMID: 24229411
11.  Comparative Efficacy of Lamivudine and Emtricitabine: A Systematic Review and Meta-Analysis of Randomized Trials 
PLoS ONE  2013;8(11):e79981.
Lamivudine and emtricitabine are considered equivalent by several guidelines, but evidence of comparable efficacy is conflicting.
We searched two databases up to June 30 2013 to identify randomized and quasi-randomized trials in which lamivudine and emtricitabine were used as part of combination antiretroviral therapy for treatment-naïve or experienced HIV-positive adult patients. We only included trials where partner drugs in the regimen were identical or could be considered to be comparable. We allowed for comparisons between tenofovir and abacavir provided the study population did not begin treatment with a viral load >100,000 copies/ml.
12 trials contributed 15 different randomized comparisons providing data on 2251 patients receiving lamivudine and 2662 patients receiving emtricitabine. Treatment success was not significantly different in any of the 12 trials. In the three trials that directly compared lamivudine and emtricitabine, the relative risk for achieving treatment success was non-significant (RR 1.03 95%CI 0.96-1.10). For all trials combined, the pooled relative risk for treatment success was not significantly different (RR 1.00, 95%CI 0.97–1.02). No heterogeneity was observed (I2 = 0). Similarly, there was no difference in the pooled relative risk for treatment failure (RR 1.08, 95%CI 0.94–1.22, I2 = 3.4%).
The findings of this systematic review suggest that lamivudine and emtricitabine are clinically equivalent.
PMCID: PMC3823593  PMID: 24244586
12.  Adherence as a Predictor of the Development of Class-Specific Resistance Mutations: The Swiss HIV Cohort Study 
PLoS ONE  2013;8(10):e77691.
Non-adherence is one of the strongest predictors of therapeutic failure in HIV-positive patients. Virologic failure with subsequent emergence of resistance reduces future treatment options and long-term clinical success.
Prospective observational cohort study including patients starting new class of antiretroviral therapy (ART) between 2003 and 2010. Participants were naïve to ART class and completed ≥1 adherence questionnaire prior to resistance testing. Outcomes were development of any IAS-USA, class-specific, or M184V mutations. Associations between adherence and resistance were estimated using logistic regression models stratified by ART class.
Of 314 included individuals, 162 started NNRTI and 152 a PI/r regimen. Adherence was similar between groups with 85% reporting adherence ≥95%. Number of new mutations increased with increasing non-adherence. In NNRTI group, multivariable models indicated a significant linear association in odds of developing IAS-USA (odds ratio (OR) 1.66, 95% confidence interval (CI): 1.04-2.67) or class-specific (OR 1.65, 95% CI: 1.00-2.70) mutations. Levels of drug resistance were considerably lower in PI/r group and adherence was only significantly associated with M184V mutations (OR 8.38, 95% CI: 1.26-55.70). Adherence was significantly associated with HIV RNA in PI/r but not NNRTI regimens.
Therapies containing PI/r appear more forgiving to incomplete adherence compared with NNRTI regimens, which allow higher levels of resistance, even with adherence above 95%. However, in failing PI/r regimens good adherence may prevent accumulation of further resistance mutations and therefore help to preserve future drug options. In contrast, adherence levels have little impact on NNRTI treatments once the first mutations have emerged.
PMCID: PMC3797701  PMID: 24147057
13.  Dolutegravir Interactions with HIV-1 Integrase-DNA: Structural Rationale for Drug Resistance and Dissociation Kinetics 
PLoS ONE  2013;8(10):e77448.
Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.
PMCID: PMC3797783  PMID: 24146996
14.  Ritonavir-Boosted Darunavir Is Rarely Associated with Nephrolithiasis Compared with Ritonavir-Boosted Atazanavir in HIV-Infected Patients 
PLoS ONE  2013;8(10):e77268.
Although ritonavir-boosted atazanavir (ATV/r) is known to be associated with nephrolithiasis, little is known about the incidence of nephrolithiasis in patients treated with ritonavir-boosted Darunavir (DRV/r), the other preferred protease inhibitor.
In a single-center cohort, the incidence of nephrolithiasis was compared between HIV-infected patients who commenced DRV/r-containing antiretroviral therapy and those on ATV/r. The effects of ATV/r use over DRV/r were estimated by univariate and multivariate Cox hazards models.
Renal stones were diagnosed in only one patient (0.86 per 1000 person-years) of the DRV/r group (n=540) and 37 (20.2 per 1000 person-years) of the ATV/r group (n=517). The median [interquartile (IQR)] observation period in the DRV/r group was 27.1 months (IQR 18.1-38.4 months), and 40.6 months (IQR 17.5-42.7) for the ATV/r group. The total observation period was 1,163.6 person-years and 1,829.6 person-years for the DRV/r group and for the ATV/r group, respectively. In the 37 patients on ATV/r who developed nephrolithiasis, the median time from commencement of ATV/r to diagnosis was 28.1 months (IQR 18.4–42.7), whereas nephrolithiasis in the single patient of the DRV/r group occurred 11.2 month after the introduction of DRV/r. ATV/r use over DRV/r was significantly associated with nephrolithiasis by uni- and multivariate analyses (HR=26.01; 95% CI, 3.541–191.0; p=0.001) (adjusted HR=21.47; 95% CI, 2.879–160.2; p=0.003).
The incidence of nephrolithiasis was substantially lower in patients on DRV/r than those on ATV/r. The results suggest that DRV/r should be selected for treatment of HIV-infected patients at risk of chronic kidney disease.
PMCID: PMC3795077  PMID: 24130871
15.  Phylogenetic inferences on HIV-1 transmission: implications for the design of prevention and treatment interventions 
AIDS (London, England)  2013;27(7):1045-1057.
PMCID: PMC3786580  PMID: 23902920
HIV-1 transmission; phylogenetics; primary infection; treatment as prevention
17.  Next-Generation Sequencing of HIV-1 RNA Genomes: Determination of Error Rates and Minimizing Artificial Recombination 
PLoS ONE  2013;8(9):e74249.
Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04–0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9–2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.
PMCID: PMC3776835  PMID: 24058534
18.  Is the HIV Epidemic Stable among MSM in Mexico? HIV Prevalence and Risk Behavior Results from a Nationally Representative Survey among Men Who Have Sex with Men 
PLoS ONE  2013;8(9):e72616.
Recent evidence points to the apparent increase of HIV prevalence among men who have sex with men (MSM) in different settings with concentrated epidemics, including the Latin American region. In 2011, Mexico implemented an ambitious HIV prevention program in all major cities, funded by the Global Fund to Fight Aids, Tuberculosis and Malaria. The program was intended to strengthen the prevention response for the most at risk populations: MSM and injecting drug users. This paper presents the HIV prevalence results of a nationally representative baseline survey in 24 Mexican cities throughout the 5 regions in the country and reports the socio-demographic and sexual risk behaviors that predict the probability of infection.
The survey was implemented in two phases. We first identified and characterized places where MSM gather in each city and then conducted in a second phase, a seroprevalence survey that included rapid HIV testing and a self-administered questionnaire. The prevalence of HIV was estimated by adjusting for positive predicted value. We applied a probit model to estimate the probability of having a positive result from the HIV test as a function of socio-demographic characteristics and self-reported sexual risk behaviors.
We found an overall HIV prevalence among MSM gathering in meeting points of 16.9% [95% CI: 15.6–18.3], significantly higher than previously reported estimates. Our regression results suggest that the risk of infection increases with age, with the number of sexual partners, and among those who play a receptive sexual role, and the risk decreases with higher education.
Our findings suggest a higher HIV prevalence among MSM than previously acknowledged and that a significant regional variability exist throughout the country. These two findings combined, signal an important dynamic in the epidemic that should be better understood and promptly addressed with strong prevention efforts targeted at key populations.
PMCID: PMC3764146  PMID: 24039786
19.  Evolving uses of oral reverse transcriptase inhibitors in the HIV-1 epidemic: from treatment to prevention 
Retrovirology  2013;10:82.
The HIV epidemic continues unabated, with no highly effective vaccine and no cure. Each new infection has significant economic, social and human costs and prevention efforts are now as great a priority as global antiretroviral therapy (ART) scale up. Reverse transcriptase inhibitors, the first licensed class of ART, have been at the forefront of treatment and prevention of mother to child transmission over the past two decades. Now, their use in adult prevention is being extensively investigated. We describe two approaches: treatment as prevention (TasP) - the use of combination ART (2NRTI and 1NNRTI) following HIV diagnosis to limit transmission and pre-exposure prophylaxis (PrEP) –the use of single or dual oral agents prior to sexual exposure. Prevention of mother-to-child transmission using NRTI has been highly successful, though does not involve sustained use of NRTI to limit transmission. Despite theoretical and preliminary support for TasP and PrEP, data thus far indicate that adherence, retention in care and late diagnosis are the major barriers to their successful, sustained implementation. Future advances in drug technologies will be needed to overcome the issue of drug adherence, through development of drugs that involve both less frequent dosing as well as reduced toxicity, possibly through specific targeting of infected cells.
PMCID: PMC3733946  PMID: 23902855
20.  Automethylation of protein arginine methyltransferase 6 (PRMT6) regulates its stability and its anti-HIV-1 activity 
Retrovirology  2013;10:73.
Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.
Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays.
These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.
PMCID: PMC3750301  PMID: 23866860
PRMT6; HIV-1; Automethylation; Protein stability; Antiretroviral activity
21.  Rates and Factors Associated with Major Modifications to First-Line Combination Antiretroviral Therapy: Results from the Asia-Pacific Region 
PLoS ONE  2013;8(6):e64902.
In the Asia-Pacific region many countries have adopted the WHO’s public health approach to HIV care and treatment. We performed exploratory analyses of the factors associated with first major modification to first-line combination antiretroviral therapy (ART) in resource-rich and resource-limited countries in the region.
We selected treatment naive HIV-positive adults from the Australian HIV Observational Database (AHOD) and the TREAT Asia HIV Observational Database (TAHOD). We dichotomised each country’s per capita income into high/upper-middle (T-H) and lower-middle/low (T-L). Survival methods stratified by income were used to explore time to first major modification of first-line ART and associated factors. We defined a treatment modification as either initiation of a new class of antiretroviral (ARV) or a substitution of two or more ARV agents from within the same ARV class.
A total of 4250 patients had 961 major modifications to first-line ART in the first five years of therapy. The cumulative incidence (95% CI) of treatment modification was 0.48 (0.44–0.52), 0.33 (0.30–0.36) and 0.21 (0.18–0.23) for AHOD, T-H and T-L respectively. We found no strong associations between typical patient characteristic factors and rates of treatment modification. In AHOD, relative to sites that monitor twice-yearly (both CD4 and HIV RNA-VL), quarterly monitoring corresponded with a doubling of the rate of treatment modifications. In T-H, relative to sites that monitor once-yearly (both CD4 and HIV RNA-VL), monitoring twice-yearly corresponded to a 1.8 factor increase in treatment modifications. In T-L, no sites on average monitored both CD4 & HIV RNA-VL concurrently once-yearly. We found no differences in rates of modifications for once- or twice-yearly CD4 count monitoring.
Low-income countries tended to have lower rates of major modifications made to first-line ART compared to higher-income countries. In higher-income countries, an increased rate of RNA-VL monitoring was associated with increased modifications to first-line ART.
PMCID: PMC3696001  PMID: 23840312
22.  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.
PMCID: PMC3497622  PMID: 22993165
23.  In Vitro HIV-1 Evolution in Response to Triple Reverse Transcriptase Inhibitors & In Silico Phenotypic Analysis 
PLoS ONE  2013;8(4):e61102.
Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance.
Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments.
Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates.
Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here “in silico” from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of “wet-” and “dry-lab” experimentation may improve our understanding of HIV-1 resistance evolution in the future.
PMCID: PMC3629221  PMID: 23613794
25.  Antiviral Drug Resistance and the Need for Development of New HIV-1 Reverse Transcriptase Inhibitors 
Antimicrobial Agents and Chemotherapy  2012;56(10):5000-5008.
Highly active antiretroviral therapy (HAART) consists of a combination of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. Despite being the first antivirals described to be effective against HIV, reverse transcriptase inhibitors remain the cornerstone of HAART. There are two broad classes of reverse transcriptase inhibitor, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first such compounds were developed, viral resistance to them has inevitably been described; this necessitates the continuous development of novel compounds within each class. In this review, we consider the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second-line therapy and describe the patterns of resistance associated with their use as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with a low genetic barrier are more commonly used in resource-limited settings. Their use results in the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings.
PMCID: PMC3457356  PMID: 22733071

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