D30N Mutation Research Articles

Outcome of Darunavir-Cobicistat-Based Regimens in HIV-Infected People Who Have Experienced Virological Failure.

To evaluate the virological outcome of darunavir-cobicistat (DRVc)-based regimens in adults living with HIV who had experienced virological failure (VF) on any previous drug combination. This was a retrospective cohort study (CSLHIV Cohort) of adults living with HIV who started a DRVc-based regimen with HIV-RNA >50 copies/mL after VF on any previous drug combination. Data on demographics, antiretroviral treatment since HIV diagnosis, and immunological and metabolic parameters from baseline (start of DRVc) to 48 weeks were analyzed in order to assess the cumulative proportion of those who achieved virological success (VS), defined as at least one instance of HIV-RNA <50 copies/mL within 12 months from baseline. Follow-up lasted from the start of the DRVc-based regimen (baseline) to the first instance of HIV-RNA <50 copies/mL, last available visit, or lossto follow-up or death, whichever occurred first. Univariate and multivariate Cox proportional-hazard regression models were used to identify baseline factors associated with VS. A total of 176 individuals were included, and 120 (68.2%) achieved <50 HIV-RNA copies/mL within 12 months since baseline. On multivariate analysis, baseline HDL cholesterol was independently associated with the occurrence of VS (adjusted HR 1.021, 95% CI 1.004-1.038; p=0.014). Among the 120 subjects with VS, 27 (22.5%) had had VF during a median follow-up of 20.8 months since the first undetectable HIV-RNA. Resistance testing after VF was available in two cases, which harboured the HIV variant-bearing protease inhibitor-resistance mutations D30N, I50V, and N88D. During a median follow-up of 38.4 months, 65 of 176 (36.9%) individuals discontinued DRVc for any reason (37 of 120, 30.8%) and achieved VS vs. 28 of 56 (50%) without VS (p=0.019). Time to discontinuation was longer in people with VS (41.5 vs. 23.0 months, p=0.0007). No statistically significant changes were observed in immunological or lipid profiles during follow-up. Most individuals in this study achieved VS within 12 months from the beginning of a DRVc-based regimen; therefore, this treatment represent a viable option for people who have experienced VF on other regimens.

Molecular basis for reduced cleavage activity and drug resistance in D30N HIV-1 protease

Nelfinavir is one of the FDA-approved HIV-1 protease inhibitors and a part of highly active anti-retroviral therapy (HAART) for the treatment of HIV-AIDS. Nelfinavir was the first HIV-1 protease inhibitor to be approved as a paediatric formulation. The application of HAART had resulted in significant improvement in the lives of AIDS patients. However, the emergence of drug resistance in HIV-1 protease has limited the use of many of these drugs including nelfinavir. A unique mutation observed frequently in patients treated with nelfinavir is D30N as it is selected exclusively by nelfinavir. The D30N mutation imparts very high resistance to nelfinavir but unlike other primary mutations does not give cross-resistance to the majority of other drugs. D30N mutation also significantly reduces cleavage activity of HIV-1 protease and affects viral fitness. Here, we have determined crystal structures of D30N HIV-1 protease in unliganded form and in complex with nelfinavir. These structures provide the rationale for reduced cleavage activity and the molecular basis of drug resistance induced by D30N mutation. The loss of coulombic interaction part of a crucial hydrogen bond between the drug and the protease is likely to play a major role in reduced affinity and resistance towards nelfinavir. The decreased catalytic activity of D30N HIV-1 protease due to altered interaction with the substrates and reduced stability of folding core may be the reason for the reduced replicative capacity of the virus harboring mutant HIV-1 protease. Communicated by Ramaswamy H. Sarma

Silent mutations at codons 65 and 66 in reverse transcriptase alleviate indel formation and restore fitness in subtype B HIV-1 containing D67N and K70R drug resistance mutations.

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or ‘silent’ mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65–67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65–67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses.

New Insights into the In Silico Prediction of HIV Protease Resistance to Nelfinavir

The Human Immunodeficiency Virus type 1 protease enzyme (HIV-1 PR) is one of the most important targets of antiretroviral therapy used in the treatment of AIDS patients. The success of protease-inhibitors (PIs), however, is often limited by the emergence of protease mutations that can confer resistance to a specific drug, or even to multiple PIs. In the present study, we used bioinformatics tools to evaluate the impact of the unusual mutations D30V and V32E over the dynamics of the PR-Nelfinavir complex, considering that codons involved in these mutations were previously related to major drug resistance to Nelfinavir. Both studied mutations presented structural features that indicate resistance to Nelfinavir, each one with a different impact over the interaction with the drug. The D30V mutation triggered a subtle change in the PR structure, which was also observed for the well-known Nelfinavir resistance mutation D30N, while the V32E exchange presented a much more dramatic impact over the PR flap dynamics. Moreover, our in silico approach was also able to describe different binding modes of the drug when bound to different proteases, identifying specific features of HIV-1 subtype B and subtype C proteases.

Naturally occurring protease inhibitor resistance mutations and their frequencies in HIV proviral sequences of drug-naïve sex workers in Nairobi, Kenya

Sub-Saharan Africa accounts for 69% of the people living with HIV globally. An estimated 1,600,000 Kenyans are living with HIV-1. Antiretroviral therapy (ART) has saved 9 million life-years in Sub-Saharan Africa. However, drug resistance mutations reduce the effectiveness of ART, and need to be monitored for effective ART. Naturally occurring primary antiretroviral drug resistance mutations have not been well analyzed in ART naive HIV+ patients in Kenya. Here we have examined protease inhibitor (PI) resistance mutations in ART naive HIV-1 seropositive women in Pumwani sex worker cohort established in Nairobi, Kenya, wherein HIV-1 infection is predominantly caused by subtypes A and D viruses. We have analyzed consensus sequences of HIV protease from 109 drug-naive patients, as a part of HIV-1 whole-genome sequencing using 454 sequencing methodology. Analysis using HIVdb program revealed a prevalence of 22% (24/109) PI resistance mutations among the study subjects. D30N (3.7%), M46I (0.9%) and V82F (0.9%) are the major mutations observed. D30N mutation is known to confer high-level resistance to nelfinavir. M46I and V82F confer resistance to indinavir, lopinavir, fosamprenavir and nelfinavir. In addition, many minor mutations were found at seven different drug resistance sites. It is important to study the implications of these mutations to the effectiveness of specific PI drug treatment. This study provides valuable data pertaining to primary drug resistance in Kenyan HIV-1 infected patients before ART became available.

Analysis of HIV‐1 primary drug resistance in Kazakhstan

Purpose of the studyMonitoring of primary resistance in HIV‐1 variants circulating in Russia and FSU countries is the important task of HIV infection molecular epidemiology. The data of HIV molecular epidemiology are absent or limited in many FSU countries. IDU‐A variant of HIV‐1 subtype A has been dominating in Russia (&gt;90%) and other FSU countries since 1996. Additionally, the Central Asian region (e.g. Kazakhstan and Uzbekistan) is characterized by relatively wide spread of CRF02_AG recombinant. The aim of our study was the analysis of HIV primary drug resistance in HIV‐1 variants from Kazakhstan.MethodsBlood collection from the HIV‐infected naïve patients was carried out by local specialists. The study was performed with the informed consent of patients. All sequences of pol gene were analyzed by COMETv0.1 and HIVdb on‐line programs. The phylogenetic analysis and tropism testing were carried out by MEGA4.0 and geno2pheno.Summary of results51 PBMC samples were analyzed. According to pol gene phylogenetic analysis and genotyping 24 (47.0%) samples belonged to recombinant CRF02_AG; 25 (49.0%)‐to subtype A1; 2 (3.9%)‐to CRF03_AB circulating in Russia and some FSU countries. Only one subtype A1 sample had D30N mutation in Pro‐region that cause high‐level resistance to NFV. All AG‐samples had K20I substitution which is characteristic for HIV‐1 subtype A and G and is believed to be associated with resistance to LPV and NFV. In addition, we found that 2 AG‐samples had L10V and L76I substitutions, accordingly. Two A1‐subtype samples studied had L10I and K43T, accordingly. A62V characteristic mutation in RT‐region was found in 12 (48%) subtype A1‐samples. One of them had M184I mutation as well. Besides, we found 3 A1‐samples harboring NNRTIs resistance mutations‐K103N, G190S, K238N, accordingly. Further we analyzed IN region of pol‐gene in 16 and env‐gene in 23 samples studied. We found only one E157Q mutation (in CRF03_AB sample) in IN region. As to tropism, only two subtype A1 and 1 CRF03_AB samples belonged to X4/X4R5 variant, all these A1‐subtype samples harbored A62V and one of them‐G190S in RT region of pol‐gene; the other samples were treated as R5 variants.ConclusionsOur data demonstrated the low level of HIV primary drug resistance in Kazakhstan. HIV‐1 subtype A variant dominates on this territory, but CRF02_AG recombinant is rather widespread as well. CRF02_AG variant studied has characteristic features in Pro region compared with IDU‐A.

Zidovudine (AZT) Monotherapy Selects for the A360V Mutation in the Connection Domain of HIV-1 Reverse Transcriptase

BackgroundWe 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 FindingsFull-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.ConclusionsThe A360V mutation in the connection domain of RT was selected in HIV-infected individuals that received AZT monotherapy and contributed to AZT resistance.

Prevalence of HIV Drug Resistance Mutations in HIV Type 1 Isolates in Antiretroviral Therapy Naïve Population from Northern India

Objective. The increased use of antiretroviral therapy (ART) has reduced the morbidity and mortality associated with HIV, adversely leading to the emergence of HIV drug resistance (HIVDR). In this study we aim to evaluate the prevalence of HIVDR mutations in ART-naive HIV-1 infected patients from northern India. Design. Analysis was performed using Viroseq genotyping system based on sequencing of entire protease and two-thirds of the Reverse Transcriptase (RT) region of pol gene. Results. Seventy three chronic HIV-1 infected ART naïve patients eligible for first line ART were enrolled from April 2006 to August 2008. In 68 patients DNA was successfully amplified and sequencing was done. 97% of HIV-1 strains belonged to subtype C, and one each to subtype A1 and subtype B. The overall prevalence of primary DRMs was 2.9% [2/68, 95% confidence interval (CI), 0.3%–10.2%]. One patient had a major RT mutation M184V, known to confer resistance to lamivudine, and another had a major protease inhibitor (PI) mutation D30N that imparts resistance to nelfinavir. Conclusion. Our study shows that primary HIVDR mutations have a prevalence of 2.9% among ART-naive chronic HIV-1 infected individuals.

RNA Interference as a Tool for Exploring HIV-1 Robustness

Short interfering RNAs (siRNAs) that target viral genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, there is the potential for viral escape, particularly with a highly mutable target such as HIV-1. We present a novel strategy for anticipating and preventing viral escape using second-generation siRNAs. The evolutionary capacity of HIV-1 was tested by exerting strong selective pressure on a highly conserved sequence in the HIV-1 genome. We assayed the antiviral efficacy of five overlapping siRNAs directed against an essential region of the HIV-1 protease. Serial viral transfers in U87-CD4-CXCR4 cells were performed using four of the siRNAs. This procedure was repeated until virus breakthrough was detected . After several serial culture passages, resistant virus with a single point mutation in the targeted region was detected in the culture supernatants. The emergence of resistant virus was confirmed by molecular cloning and DNA sequencing of viral RNA. The most common escape route was the D30N mutation. Importantly, the addition of a second-generation siRNA that matched the D30N mutation restored viral inhibition and delayed development of escape variants. Passages performed with both siRNAs prevented the emergence of the D30N escape mutant and forced the virus to develop new escape routes. Thus, second-generation siRNAs can be used to block escape from RNA interference (RNAi) and to search for new RNAi escape routes. The protocol described here may be useful for exploring the sequence space available for HIV-1 evolution and for producing attenuated or deleterious viruses.

The impact of the nelfinavir resistance-conferring mutation D30N on the susceptibility of HIV-1 subtype B to other protease inhibitors

The human immunodeficiency virus type 1 (HIV-1) protease mutation D30N is exclusively selected by the protease inhibitor (PI) nelfinavir and confers resistance to this drug. We demonstrate that D30N increases the susceptibility to saquinavir (SQV) and amprenavir in HIV-1 subtype B isolates and that the N88D mutation in a D30N background neutralizes this effect. D30N also suppresses indinavir (IDV) resistance caused by the M46I mutation. Interestingly, in patients with viruses originally containing the D30N mutation who were treated with IDV or SQV, the virus either reversed this mutation or acquired N88D, suggesting an antagonistic effect of D30N upon exposure to these PIs. These findings can improve direct salvage drug treatment in resource limited countries where subtype B is epidemiologically important and extend the value of first and second line PIs in these populations.

Double Electron-Electron Resonance and 1H-15N HSQC Spectral Analysis as Means to Understand the Effects of Single and Multiple Site Mutations in the HIV-1 protease

In this study, site-directed spin labeling coupled with electron paramagnetic resonance (SDSL-EPR) was used to probe changes in the conformational sampling of subtype B human immunodeficiency virus type 1 protease (HIV-1PR) attributed to the active site mutation D30N and the non-active site mutations M36I and A71V. Double electron-electron resonance (DEER) was employed to derive distance distribution profiles for each MTSL-labeled construct. Those with double mutations (D30N M36I and D30N A71V) exhibited higher percentage of the closed conformation while that with triple mutation (D30N M36I A71V) has a higher percentage of wide-open conformation relative to wild-type. The percentage semi-open conformation markedly decreased in the former constructs while it is comparable to wild-type for the latter.Meanwhile, 1H-15N heteronuclear single quantum coherence (HSQC) spectra were obtained for HIV-1PR constructs with single (M36I), double (D30N M36I) and triple mutations (D30N M36I A71V), and were compared to that of the wild-type. HIV-1PR residues that have marked chemical shift perturbations as a result of an active or non-active site mutation were identified. Non-active site mutations occurring at the flap elbow (M36I) or beta-sheet core (A71V) of the protease were found to affect chemical shift more than the mutation at the active site pocket (D30N). Information gained from single-point mutation-induced changes in the conformational sampling and dynamics of the HIV-1PR may provide key insights on the mechanism of drug resistance.

The HIV Type 1 Protease L10I Minor Mutation Decreases Replication Capacity and Confers Resistance to Protease Inhibitors

The effect of minor mutations in PR on treatment outcome has not been well established. We characterized the HIV protease minor mutations, L10I, compared to the minor mutation, L63P, and the major mutation D30N and their impact on viral fitness and resistance to protease inhibitors. Mutations were introduced individually and in combination by site-directed mutagenesis into the provirus pNL4.3ren and constructs used for replication capacity (RC) and resistance assays. A structure prediction of the protease carrying the L10I mutation was determined. The prevalence of the minor mutation L10I had a pattern similar to that found for major mutations D30N, with a low prevalence (4.9%) in naive patients and significantly higher prevalence in treated patients. Furthermore, viruses carrying the major mutation D30N or the minor mutation L10I showed a significant decrease in RC (p-value <0.05), whereas viruses carrying the minor mutation L63P had RC similar to wild-type virus. In addition, the L10I mutation conferred resistance to saquinavir, which was supported by the higher prevalence in the cohort of the L10I mutation among patients with SQV resistance. The molecular modeling suggests that L10I may affect the conformation of Leu-23, a critical residue in the substrate binding site. In conclusion, the L10I mutation impairs RC and confers resistance to SQV, similarly to other major mutations, which may be related with changes in the conformation in the protease binding site. The presence of this mutation in the genotype of HIV from patients should be taken into consideration when designing new optimize treatments.

In Vivo validation of a bioinformatics based tool to identify reduced replication capacity in HIV-1.

Although antiretroviral drug resistance is common in treated HIV infected individuals, it is not a consistent indicator of HIV morbidity and mortality. To the contrary, HIV resistance-associated mutations may lead to changes in viral fitness that are beneficial to infected individuals. Using a bioinformatics-based model to assess the effects of numerous drug resistance mutations, we determined that the D30N mutation in HIV-1 protease had the largest decrease in replication capacity among known protease resistance mutations. To test this in silico result in an in vivo environment, we constructed several drug-resistant mutant HIV-1 strains and compared their relative fitness utilizing the SCID-hu mouse model. We found HIV-1 containing the D30N mutation had a significant defect in vivo, showing impaired replication kinetics and a decreased ability to deplete CD4+ thymocytes, compared to the wild-type or virus without the D30N mutation. In comparison, virus containing the M184V mutation in reverse transcriptase, which shows decreased replication capacity in vitro, did not have an effect on viral fitness in vivo. Thus, in this study we have verified an in silico bioinformatics result with a biological assessment to identify a unique mutation in HIV-1 that has a significant fitness defect in vivo.

Resistant mechanism against nelfinavir of subtype C human immunodeficiency virus type 1 proteases

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery, and the subtype C HIV-1 is infecting more and more humans. In this work, we executed computational simulations of subtype B (labeled B(WT)) HIV-1 PR, D30N mutant B (labeled B(D30N)) HIV-1 PR, subtype C (labeled C(Ref)) HIV-1 PR, D30N mutant C (labeled C(D30N)) HIV-1 PR, and D30N/N83T mutant C (labeled C(D30N/N83T)) HIV-1 PR with drug nelfinavir (NFV), aiming at clarifying (1) the resistant mechanism against NFV due to D30N mutation in subtype C HIV-1 PR; (2) the reason that the emergence rate of N83T mutation in C(D30N) HIV-1 PR is higher than that in B(D30N) HIV-1 PR; (3) the affinity of NFV with C(D30N/N83T) HIV-1 PR is higher than B(D30N) and C(D30N) HIV-1 PRs. The results indicate: (1) D30N mutation in subtype C HIV-1 PR reduces the hydrogen bond between the 30th residue and NFV, and the binding free energy contributions of some residues decrease; (2) the hydrogen bonds between the 83th/83′th residue and the 34th/34′th residue exist in both B(D30N) and C(D30N/N83T) HIV-1 PRs, while they disappear in C(D30N) HIV-1 PR. Meanwhile, the binding free energy contribution of N30 in C(D30N) is lower than that in B(D30N) and C(D30N/N83T); (3) N83T mutation makes some residues dislocate, and the contributions of these residues to binding free energy in C(D30N/N83T) increase comparing to those in B(D30N) and C(D30N). Our findings suggest that despite the nonactive site mutations, the polymorphisms regulate the emergence rates of these drug-resistant mutants.

The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01_AE (AE) strain is seen principally in Southeast Asia. AE protease differs by approximately 10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.

Understanding the HIV-1 protease nelfinavir resistance mutation D30N in subtypes B and C through molecular dynamics simulations

A major concern in the antiretroviral (ARV) treatment of HIV infections with protease inhibitors (PI) is the emergence of resistance, which results from the selection of distinct mutations within the viral protease (PR) gene. Among patients who do not respond to treatment with the PI nelfinavir (NFV), the D30N mutation is often observed. However, several reports have shown that D30N emerges with different frequencies in distinct HIV-1 genetic forms or subtypes. In the present work, we analyzed the binding of NFV and the Gag substrate CA/p2 to PR from HIV-1 subtypes B and C through molecular dynamics (MD) simulations. The wild-type and drug-resistant D30N mutants were investigated in both subtypes. The compensatory mutations N83T and N88D, observed in vitro and in vivo when subtype C acquires D30N, were also studied. D30N appears to facilitate conformational changes in subtype B PR, but not in that from subtype C, and this could be associated with disestablishment of an α-helical region of the PR. Furthermore, the total contact areas of NFV or the CA/p2 substrate with the mutant PR correlated with changes in the resistance patterns and replicative capacity. Finally, we observed in our MD simulations that mutant PR proteins show different patterns for hydrophobic/van der Waals contact. These findings suggest that different molecular mechanisms contribute to resistance, and we propose that a single mutation has distinct impacts on different HIV-1 subtypes.

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.

Postpartum antiretroviral drug resistance in HIV-1-infected women receiving pregnancy-limited antiretroviral therapy.

Pregnancy-limited antiretroviral therapy (PLAT) drastically reduces HIV-1 transmission to the newborn, but may select for antiretroviral drug resistance mutations in mothers. We evaluated antiretroviral-naive, HIV-1-infected pregnant women who received PLAT between 1998 and 2005, and had 2-month or 6-month postpartum plasma samples available with HIV-1 RNA levels more than 500 copies/ml. Postpartum drug resistance mutation rates were assessed blindly using population sequencing and allele-specific PCR (ASPCR) of the M184V, K103N and D30N mutations. Factors associated with selection of drug resistance mutations were investigated. One hundred and forty-six women were included. All women received zidovudine and lamivudine during pregnancy; 76% also received nelfinavir and 8.2% nevirapine. Resistance data were available from 114 women (78%). Postpartum rates of single-class, dual-class, and triple-class resistance were, respectively, 43, 6.1 and 0% (63.2, 10.5 and 1.7% by ASPCR). In women receiving dual or triple PLAT, respectively, postpartum M184V/I rates were 65% (95% by ASPCR) and 28.7% (51.6% by ASPCR), respectively (P < 0.01). Postpartum nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance rates among women receiving nevirapine were 25% for K103N (37.5% by ASPCR) and 12.5% for Y188C. Protease inhibitor resistance rates in women receiving nelfinavir were 1.1% for D30N (1.1% by ASPCR) and 1.1% for L90M. Dual versus triple PLAT and prolonged zidovudine exposure were associated with selection of M184V. Nevirapine use and length of zidovudine and lamivudine exposure were associated with selection of K103N. PLAT is associated with frequent selection of resistance to drugs with low-genetic barrier. Triple-drug PLAT decreases the odds for M184V selection. Routine postpartum genotypic resistance testing may be useful to guide future treatment decisions in mothers.

Structural and Energetic Analysis on the Complexes of Clinically Isolated Subtype C HIV-1 Proteases and Approved Inhibitors by Molecular Dynamics Simulation

HIV-1 has a large genetic diversity. Subtype B HIV-1 is commonly found in patients in developed countries. In contrast, an increasing number of patients are infected with the non-B subtype viruses, especially with subtype C HIV-1, in developing countries. It remains to be clarified how mutations or polymorphisms in non-B subtype HIV-1 influence the efficacy of the approved inhibitors. In this study, we have performed molecular dynamics simulations on clinically isolated subtype C HIV-1 proteases in complex with three kinds of approved inhibitors. From the structural and energetic viewpoints, we identified the polymorphisms influencing on the binding of the inhibitors. The effect of the V82I mutation on the association with chemicals and the reason for rare appearance of the D30N mutation in subtype C HIV-1 were discussed in terms of the change of geometry of the residues in HIV-1 protease.

Insights into drug resistance of mutations D30N and I50V to HIV-1 protease inhibitor TMC-114: Free energy calculation and molecular dynamic simulation

The single mutations D30N and I50V are considered as the key residue mutations of the HIV-1 protease drug resistance to inhibitors in clinical use. In this work, molecular dynamics (MD) simulations combined with the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method have been performed to investigate the drug-resistant mechanisms of D30N and I50V to an inhibitor TMC-114. The analyses of absolute binding free energies using the separate trajectory approach suggests that the decrease in the van der Waals energy and electrostatic energy in the gas phase results in the drug resistance of D30N to TMC-114, while for I50V, the decrease in the electrostatic energy mainly drive its drug resistance to TMC-114. Detailed binding free energies between TMC-114 and individual protein residues are computed by using a per-residue basis decomposition method, which provides insights into the inhibitor-protein binding mechanism and also explains the drug-resistant mechanisms of mutations D30N and I50V to TMC-114. The study shows that the loss of the hydrogen bond between TMC-114 and the side chain of Asn30' is the main driving force of the resistance of D30N to TMC-114, and in the case of I50V, the increase in the polar solvation energies between TMC-114 and two residues Val50' and Asp30' definitively drives the resistance of I50V to TMC-114. We expect that this work can provide some helpful insights into the nature of mutational effect and aid the future design of better inhibitors.