Wide variation in susceptibility of transmitted/founder HIV-1 subtype C Isolates to protease inhibitors and association with in vitro replication efficiency

© 2016 The Author(s).The gag gene is highly polymorphic across HIV-1 subtypes and contributes to susceptibility to protease inhibitors (PI), a critical class of antiretrovirals that will be used in up to 2 million individuals as second-line therapy in sub Saharan Africa by 2020. Given subtype C represents around half of all HIV-1 infections globally, we examined PI susceptibility in subtype C viruses from treatment-naïve individuals. PI susceptibility was measured in a single round infection assay of full-length, replication competent MJ4/gag chimeric viruses, encoding the gag gene and 142 nucleotides of pro derived from viruses in 20 patients in the Zambia-Emory HIV Research Project acute infection cohort. Ten-fold variation in susceptibility to PIs atazanavir and lopinavir was observed across 20 viruses, with EC50 s ranging 0.71-6.95 nM for atazanvir and 0.64-8.54 nM for lopinavir. Ten amino acid residues in Gag correlated with lopinavir EC50 (p < 0.01), of which 380 K and 389I showed modest impacts on in vitro drug susceptibility. Finally a significant relationship between drug susceptibility and replication capacity was observed for atazanavir and lopinavir but not darunavir. Our findings demonstrate large variation in susceptibility of PI-naïve subtype C viruses that appears to correlate with replication efficiency and could impact clinical outcomes

Gag-protease coevolution analyses define novel structural surfaces in the HIV-1 matrix and capsid involved in resistance to Protease Inhibitors

Despite the major role of Gag in establishing resistance of HIV-1 to protease inhibitors (PIs), very limited data are available on the total contribution of Gag residues to resistance to PIs. To identify in detail Gag residues and structural interfaces associated with the development of HIV-1 resistance to PIs, we traced viral evolution under the pressure of PIs using Gag-protease single genome sequencing and coevolution analysis of protein sequences in 4 patients treated with PIs over a 9-year period. We identified a total of 38 Gag residues correlated with the protease, 32 of which were outside Gag cleavage sites. These residues were distributed in 23 Gag-protease groups of coevolution, with the viral matrix and the capsid represented in 87% and 52% of the groups. In addition, we uncovered the distribution of Gag correlated residues in specific protein surfaces of the inner face of the viral matrix and at the Cyclophilin A binding loop of the capsid. In summary, our findings suggest a tight interdependency between Gag structural proteins and the protease during the development of resistance of HIV-1 to PIs

HIV-1 Gag mutations alone are sufficient to reduce darunavir susceptibility during virological failure to boosted PI therapy

Virological failure (VF) to boosted PIs with a high genetic barrier is not usually linked to the development of resistance-associated mutations in the protease gene. From a cohort of 520 HIV-infected subjects treated with lopinavir/ritonavir or darunavir/ritonavir monotherapy, we retrospectively identified nine patients with VF. We sequenced the HIV-1 Gag-protease region and generated clonal virus from plasma samples. We characterized phenotypically clonal variants in terms of replicative capacity and susceptibility to PIs. Also, we used VESPA to identify signature mutations and 3D molecular modelling information to detect conformational changes in the Gag region. All subjects analysed harboured Gag-associated polymorphisms in the absence of resistance mutations in the protease gene. Most Gag changes occurred outside Gag cleavage sites. VESPA analyses identified K95R and R286K (P < 0.01) as signature mutations in Gag present at VF. In one out of four patients with clonal analysis available, we identified clonal variants with high replicative capacity and 8- to 13-fold reduction in darunavir susceptibility. These clonal variants harboured K95R, R286K and additional mutations in Gag. Low susceptibility to darunavir was dependent on the Gag sequence context. All other clonal variants analysed preserved drug susceptibility and virus replicative capacity. Gag mutations may reduce darunavir susceptibility in the absence of protease mutations while preserving viral fitness. This effect is Gag-sequence context dependent and may occur during boosted PI failure

On the entropy of protein families

Proteins are essential components of living systems, capable of performing a huge variety of tasks at the molecular level, such as recognition, signalling, copy, transport, ... The protein sequences realizing a given function may largely vary across organisms, giving rise to a protein family. Here, we estimate the entropy of those families based on different approaches, including Hidden Markov Models used for protein databases and inferred statistical models reproducing the low-order (1-and 2-point) statistics of multi-sequence alignments. We also compute the entropic cost, that is, the loss in entropy resulting from a constraint acting on the protein, such as the fixation of one particular amino-acid on a specific site, and relate this notion to the escape probability of the HIV virus. The case of lattice proteins, for which the entropy can be computed exactly, allows us to provide another illustration of the concept of cost, due to the competition of different folds. The relevance of the entropy in relation to directed evolution experiments is stressed.Comment: to appear in Journal of Statistical Physic

Deep sequencing of protease inhibitor resistant HIV patient isolates reveals patterns of correlated mutations in Gag and protease.

While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle

Estudo da diversidade de vírus da imunodeficiência humana tipo 1 em indivíduos infetados de naturalidade Guineense e residentes em Portugal

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016O vírus da imunodeficiência humana (VIH) é caraterizado por uma elevada diversidade genética que resulta principalmente das taxas de mutação e recombinação genética que origina novas de variantes genómicas ao longo do tempo. Este facto, juntamente com o aparecimento de mutações de resistência aos antirretrovirais tem causado diversos problemas ao nível do diagnóstico e tratamento da infeção. O presente estudo pretendeu conhecer a diversidade genética do VIH-1 num grupo de 42 mulheres de naturalidade Guineense e residentes em Portugal e comparar os resultados com os obtidos num estudo anteriormente desenvolvido em indivíduos nativos e residentes na Guiné-Bissau. Para caracterizar as amostras foram amplificadas por nested-PCR as regiões env, nef, PR e RT do VIH-1, posteriormente sequenciadas e realizada a análise filogenética das sequências com recurso a diferentes programas bioinformáticos. Foi realizada a análise das sequências aminoacídicas das quatro regiões genómicas, observando o grau de conservação e disrupção dos principais domínios estruturais e funcionais das proteínas, e ainda, a deteção das mutações de resistência associadas aos inibidores da PR e RT do VIH-1. No grupo estudado foi obtida uma prevalência da CRF02_AG de 82% e identificados 5% de casos de infeção pelo sub-subtipo A1 (subtipo puro). Duas sequências foram classificadas em subtipo A, no entanto, limitado apenas à região env do VIH-1. Em três sequências foram observadas, diferentes recombinações (CRF02_AG/A1; CRF02_AG/J/A; 09_cpx/A) sendo classificadas como potencialmente formas recombinantes únicas. Foi ainda observado, e termos gerais uma conservação dos domínios funcionais e estruturais das proteínas. Foi identificada elevada percentagem de polimorfismos naturais, associados a subtipos não-B. Foram detetadas mutações de resistência em 16% das sequências analisadas, associadas tanto aos inibidores da protease (V11I, T74P e N88T) como aos inibidores da transcriptase reversa (A98G, K103N e M184V). Neste estudo, observou-se uma diversidade genética do VIH-1 semelhante à encontrada no estudo realizado em amostras de nativos e residentes na Guiné-Bissau e bastante inferior à diversidade descrita em Portugal. No entanto, esta diversidade de contribui para um padrão molecular da infeção no nosso país, único entre os países Europeus.The human immunodeficiency vírus (HIV) is characterized by a wide genetic diversity from higher rates of mutation and genetic recombination, originating over time, large number of genomic variants. This fact, together with the appearance of the virus with resistance mutations to the antiretroviral has caused many problems for the diagnosis, infection treatment and drug development. The present study aims to determine the genetic diversity of HIV-1 in a group of 42 individuals of Guinean nationality and residents in Portugal, comparing the results with a study developed in the laboratory with native and resident individuals of Guinea -Bissau. To characterize the samples, were amplified by nested-PCR the genomic regions, env, nef, PR and RT of the HIV-1, subsequently sequenced and held phylogenetic analysis of the sequences using different bioinformatic programs. Was performed an analyses of the amino acid sequences of the four regions of the study, observing the retention degree and disruption of the main structural and functional domains of HIV-1, and also the detection of the resistance mutations associated with the PR and RT inhibitors. In the studied group was observed a prevalence of the CRF02_AG in 82% and identified 5% of cases of infection by the sub-subtype A1 (pure subtype). Two sequences were classified as subtype A, however only limited to the env region of HIV-1. In three sequences were observed different recombinations (CRF02_AG/A1; CRF02_AG/J/A; 09_cpx/A) being classified as potentially unique recombinant forms, having a mosaic genome. It was also observed, in general, a conservation of functional and structural domains of proteins. It was identified high percentage of polymorphisms associated with non-B subtypes. Resistance mutations were detected in 16% of analysed sequences associated with both protease inhibitors (V11I, T74P and N88T) as the reverse transcriptase inhibitors (A98G, K103N and M184V). In this study, we observed a genetic diversity of HIV-1 similar to that found in the study conducted on samples of native and resident Guinea-Bissau and much lower than the diversity described in Portugal. However, this diversity contributes to a molecular pattern of infection in our country, unique among European countrie

Variabilidad natural en los sitios de procesamiento viral y en posiciones asociadas a resistencias antirretrovirales de la proteasa, retrotranscriptasa e integrasa en las variantes del VIH-1

El virus de la inmunodeficiencia humana tipo 1 (VIH-1) presenta alta variabilidad genética, debida a su alta tasa de replicación, de mutación y de recombinación, describiéndose numerosas variantes. Basándose en la homología genética de sus secuencias, el VIH-1 se clasifica en cuatro grupos: M (principal), O (outlier), N (non-M, non-O) y P. A su vez, el grupo M se subdivide en 9 subtipos (A, B, C, D, F, G, H, J, K), en al menos 88 recombinantes circulantes (CRF), y múltiples recombinantes únicos. Los recombinantes se están expandiendo en la pandemia (incluido en Europa), principalmente en lugares donde circulan simultáneamente numerosas variantes diferentes. El subtipo B es la variante predominante en Europa y Estados Unidos, pero los subtipos del VIH-1 distintos al subtipo B y recombinantes (variantes no-B) causan cerca del 90% de los 37 millones de infecciones por VIH..

Investigating the role of Gag in protease inhibitor susceptibility amongst West African HIV-1 subtypes

HIV-1 Gag contributes to susceptibility of protease inhibitors (PIs) in the absence of known resistance mutations in the protease gene. For the majority of HIV-infected patients worldwide, PIs are the second, and last-line of therapy. Clinically, only around 20% of individuals who fail PI regimen develop major resistance mutations in protease. We previously showed that full-length Gagprotease-derived phenotypic susceptibility to PIs differed between HIV-1 CRF02_AG and subtype G-infected patients who went on to successfully suppress viral replication versus those who experienced virological failure of boosted lopinavir monotherapy as first-line treatment in a clinical trial. We hypothesised therefore that baseline PI susceptibility by Gag-protease phenotyping could be used to predict treatment outcomes for patients on second line, boosted-PI treatment in the real-world clinical setting in Nigeria, where subtypes CRF02_AG/G dominate the epidemic. We used clinical and demographic data; HIV-1subtype, sex, age, viral load, duration of treatment and baseline CD4 count to match individuals who experienced second-line failure with ritonavir-boosted PI-based ART (‘baseline failures’) to those who achieved virological response (‘baseline successes’) with virological failure defined by viral load <400 copies of HIV-1 RNA/mL by week 48. Using a single replication-cycle assay, we carried out in vitro phenotypic susceptibility testing of patient-derived viruses from these two groups. We found no impact of baseline HIV-1 Gagprotease-derived phenotypic susceptibility on outcomes of PI-based second-line ART, treatment outcome could not be predicted using baseline susceptibility alone. Secondly, we sought to explore the role of mutation in Gag-protease genotypic and phenotypic changes within patients who failed PI-based regimens without known drug resistance-associated protease mutations in order to identify novel determinants of PI resistance. We used longitudinal samples collected at baseline, and at virological failure to explore the role of Gag mutations. Using target enrichment and next-generation sequencing (NGS), followed by haplotype reconstruction and phenotypic drug assays and phylogenetic analysis, we reported for the first time a four-amino acid mutation signature in HIV-1, CRF02_AG matrix (S126del, H127del, T122A and G123E) which confer reduced susceptibility to the PI, lopinavir and atazanavir. Our multi-pronged genotypic and phenotypic approach to document emergence and temporal dynamics of a novel protease inhibitor resistance signature in HIV- 1 matrix domain reveals the interplay between Gag associated resistance and fitness

Acquired and transmitted drug resistance in HIV-1 subtype C : implications of novel mutations on replication capacity, cleavage and drug susceptibility.

Doctor of Philosophy in Virology. University of KwaZulu-Natal, Medical School 2015.Introduction Large scale roll-out of combination antiretroviral therapy (cART) has been successful in improving the quality of life of HIV-1 infected individuals in South Africa (SA). However the development and transmission of drug resistance threatens the future success and longevity of cART in the country. Studies have shown that resistance to Protease inhibitors (PI’s), in the absence of mutations in Protease (PR), is increasing in SA. Whilst some studies attribute this to poor treatment adherence, others have shown that mutations in Gag contribute to PI resistance. The majority of these studies however have been conducted on HIV-1 subtype B, despite HIV-1 subtype C being the most prevalent subtype globally. Given that Gag is highly polymorphic between subtypes, studies focusing on HIV-1 subtype C are required. Despite the high rate of virologic failure of patients on PI inclusive treatment regimens, no transmitted drug resistance (TDR) studies have identified PI associated TDR mutations. This could be due to the high fitness cost associated with PR mutations which would result in rapid reversion or low frequency of mutations within the viral quasispecies. Most TDR studies in SA, as in other resource limited settings, have used recently infected cohorts to measure TDR. It is however unlikely that rapidly reverting mutations would be detected in recent infection. Furthermore, these studies have all used Sanger sequencing which only detects mutations at frequencies >15-20%. With recent studies showing that low frequency mutations present at frequencies as low as 1% impact treatment outcomes, the elucidation of these mutations using deep-sequencing techniques is necessary. For a true measure of TDR, studies employing acute infection cohorts and deep-sequencing techniques are required. The current study aimed to identify mutations in Gag-Protease associated with PI resistance/exposure, and to determine their impact on replication capacity and drug susceptibility. The prevalence of low frequency TDR mutations in an HIV-1 subtype C acute infection cohort was also investigated. Methods A cohort of 80 HIV-1 subtype C infected participants failing a PI inclusive treatment regimen (i.e. PCS cohort) from 2009–2013 in Durban, South Africa was used to assess the role of Gag in PI resistance. Gag mutations were divided into three groups: PI exposure associated Gag mutations; resistance associated Gag mutations (rGag) and novel Gag mutations (nGag). Frequencies of each of these mutations were compared amongst: 80 PCS cohort sequences, 2,481 HIV-1 subtype B treatment naïve sequences, 954 HIV-1 subtype C treatment naïve sequences and 54 HIV-1 subtype C sequences from acutely infected individuals, in order to identify PI associated mutations and natural polymorphisms. Next, recombinant viruses for all 80 participants were generated by co-transfection of a CEM derived T-cell line (i.e. GXR cells) with an NL43-deleted-gag-protease (NL43Δgag-protease) backbone and patient derived Gag-Protease amplicons. Thereafter, the replication capacity of each virus was assessed using a replication assay that employed a green fluorescent protein reporter cell line and flow cytometry. Associations between replication capacity and Gag-Protease mutations were established. Eighteen viruses with mutations of interest were then selected for use in drug susceptibility assays, where the impact of mutations on susceptibility to lopinavir (LPV) and darunavir (DRV) was assessed in a luciferase based assay. Lastly, the impact of novel Gag mutations on replication capacity and drug susceptibility was validated by generating site-directed mutant viruses with mutations of interest and using these mutant viruses in replication capacity and drug susceptibility assays. Furthermore the cleavage profile of each site-directed mutant virus was established by western blotting. Samples available from 47 HIV-1 subtype C acutely infected individuals collected from 2007-2014 in Durban, South Africa, was used to assess low frequency TDR mutations in HIV-1 subtype C acute infection. Firstly the RT and PR region of each virus was genotyped using the Viroseq HIV-1 genotyping system in order to identify the prevalence of TDR in the cohort. Thereafter 14 participant samples were selected, based on the availability of plasma at one week after onset of plasma viremia (OPV), for sequencing by ultra-deep pyrosequencing (UDPS). This served to identify low frequency mutations. Comparisons in TDR prevalence was made between Sanger sequencing and UDPS. Thereafter, the impact of low frequency TDR mutations on treatment outcomes was assessed by comparing time to virologic suppression for two participants with low frequency mutations to that of four participants without low frequency mutations. Results Protease resistance associated mutations (RAMs) occurred in 34/80 (42.5%) participants, whilst Gag mutations associated with PI resistance in subtype B were detected in 67/80 (84%) participants. Overall, 12 Gag mutations associated with PI exposure (i.e. E12K, V35I, G62R, V370A/M, S373P/Q/T, A374P, T375N, I376V, G381S, I389T, I401T and H219Q), eight rGag mutations (i.e. R76K, Y79F, V128I, A431V, K436R, L449F, R452K and P453L) and four nGag mutations (i.e. Q69K, S111C/I, T239A/S and I256V) were identified in the PCS cohort. The E12K, V370A/M, T375N, G381S, R76K and Y79F mutations all occurred as natural polymorphism in HIV-1 subtype C. The A431V, K436R, L449F, R452K, P453L, Q69K, S111C/I, T239A/S and I256V mutations were all associated with PI resistance/exposure. Interestingly all viruses with PR RAMs harboured rGag and nGag mutations, however rGag and nGag mutations were also found to occur without PR RAMs. Protease RAMs were associated with significantly reduced replication capacity. The K335R and A431V mutations were the only Gag mutations associated with significantly reduced replication capacity. Viruses with PR RAMs were associated with significantly reduced susceptibility to LPV (>15 FC in IC50) and DRV (>6 FC in IC50). Furthermore, the following combinations of rGag and nGag mutations were found to confer reduced susceptibility to LPV and DRV in the absence of PR RAMs: R76K+Y79F+K436R+L449P+I256V (5.2 fold increase in IC50 for DRV), R76K+R453L (23.88 fold increase in IC50 for LPV and a 6.73 fold increase in IC50 for DRV) and R76K+K436R+Q69K+S111C (7.40 fold increase in IC50 for LPV). Analysis of recombinant viruses showed that the Q69K nGag mutation rescued replication capacity of all viruses harbouring A431V+PR RAMs. This was validated by SDM, where Q69K rescued the replication capacity of site-directed mutant viruses harbouring A431V+V82A. The Q69K mutation was also associated with increasing polyprotein cleavage when found in conjunction with A431V+V82A. With regards to TDR, we demonstrated a prevalence of 57% of TDR mutations with UDPS and 2.2% with Sanger sequencing. Sanger sequencing identified the K103N non-nucleoside reverse transcriptase inhibitor (NNRTI)-associated TDR mutation. In addition to K103N (frequency: >99%), the following low frequency mutations were detected by UDPS: the K65R (1-1.5%) and D67N (3.88%) nucleotide reverse transcriptase inhibitor (NRTI)-associated TDR mutations, the F53L (17.6%) and M46L (6.3%) Protease inhibitor (PI)-associated TDR mutations, and the T97A (2.90%) integrase strand transfer inhibitor (InSTI)-associated TDR mutations. Participants with low frequency TDR mutations took 40 days longer to achieve viral suppression than participants without low frequency TDR mutations, when placed on fixed dose combination antiretroviral therapy