Chris M. Parry

Gag Determinants of Fitness and Drug Susceptibility in Protease Inhibitor-Resistant Human Immunodeficiency Virus Type 1

ABSTRACT Mutations can accumulate in the protease and gag genes of human immunodeficiency virus in patients who fail therapy with protease inhibitor drugs. Mutations within protease, the drug target, have been extensively studied. Mutations in gag have been less well studied, mostly concentrating on cleavage sites. A retroviral vector system has been adapted to study full-length gag, protease, and reverse transcriptase genes from patient-derived viruses. Patient plasma-derived mutant full-length gag, protease, and gag-protease from a multidrug-resistant virus were studied. Mutant protease alone led to a 95% drop in replication capacity that was completely rescued by coexpressing the full-length coevolved mutant gag gene. Cleavage site mutations have been shown to improve the replication capacity of mutated protease. Strikingly, in this study, the matrix region and part of the capsid region from the coevolved mutant gag gene were sufficient to achieve full recovery of replication capacity due to the mutant protease, without cleavage site mutations. The same region of gag from a second, unrelated, multidrug-resistant clinical isolate also rescued the replication capacity of the original mutant protease, suggesting a common mechanism that evolves with resistance to protease inhibitors. Mutant gag alone conferred reduced susceptibility to all protease inhibitors and acted synergistically when linked to mutant protease. The matrix region and partial capsid region of gag sufficient to rescue replication capacity also conferred resistance to protease inhibitors. Thus, the amino terminus of Gag has a previously unidentified and important function in protease inhibitor susceptibility and replication capacity.

Full-length HIV-1 Gag determines protease inhibitor susceptibility within in vitro assays.

Objective:There is evidence that gag contributes to protease inhibitor susceptibility in treatment-experienced patients. Moreover, protease inhibitor resistance-associated mutations can arise in gag in the absence of protease mutations in vitro. We wished to assess the contribution of full-length Gag to protease inhibitor susceptibility in viruses unexposed to protease inhibitors, in particular from the most common HIV-1 subtypes, namely subtype A and C. Design:We compared the drug resistance profiles of subtype A and C cognate gag–protease (from viruses not previously exposed to protease inhibitor) to protease combined with a generic subtype B gag as in routine phenotypic testing. Methods:We amplified gag–protease sequences from plasma-derived virus or molecular clones, and used a single cycle transfection-based drug resistance assay to compare the fold changes in the concentration of drug required to inhibit 50% of viral replication of these viruses to a generic subtype B. We made a series of chimeras to explore phenotypes further. Results:In some cases, use of protease sequences without the cognate gag overestimated susceptibility to protease inhibitors, in particular to lopinavir. We provide evidence that gag sequences from wild-type viruses can contribute as much as 14-fold reduction in susceptibility to lopinavir, and that cognate protease can balance this by partially restoring susceptibility. Conclusion:Our findings demonstrate the importance of considering protease inhibitor susceptibility in the context of full-length gag, particularly with respect to the range of HIV-1 subtypes circulating worldwide.

Three Residues in HIV-1 Matrix Contribute to Protease Inhibitor Susceptibility and Replication Capacity

ABSTRACT Other than cleavage site mutations, there is little data on specific positions within Gag that impact on HIV protease inhibitor susceptibility. We have recently shown that non-cleavage site mutations in gag, particularly within matrix protein can restore replication capacity and further reduce protease inhibitor drug susceptibility when coexpressed with a drug-resistant (mutant) protease. The matrix protein of this patient-derived virus was studied in order to identify specific changes responsible for this phenotype. Three amino acid changes in matrix (R76K, Y79F, and T81A) had an impact on replication capacity as well as drug susceptibility. Introduction of these three changes into wild-type (WT) matrix resulted in an increase in the replication capacity of the protease mutant virus to a level similar to that achieved by all the changes within the mutant matrix and part of the capsid protein. Pairs of changes to wild-type matrix led to an increased replication capacity of the protease mutant (although less than with all three changes). Having only these three changes to matrix in a wild-type virus (with wild-type protease) resulted in a 5- to 7-fold change in protease inhibitor 50% effective concentration (EC50). Individual changes did not have as great an effect on replication capacity or drug susceptibility, demonstrating an interaction between these positions, also confirmed by sequence covariation analysis. Molecular modeling predicts that each of the three mutations would result in a loss of hydrogen bonds within α-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings.

Impact of Drug Resistance-Associated Amino Acid Changes in HIV-1 Subtype C on Susceptibility to Newer Nonnucleoside Reverse Transcriptase Inhibitors

ABSTRACT The objective of this study was to assess the phenotypic susceptibility of HIV-1 subtype C isolates, with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated amino acid changes, to newer NNRTIs. A panel of 52 site-directed mutants and 38 clinically derived HIV-1 subtype C clones was created, and the isolates were assessed for phenotypic susceptibility to etravirine (ETR), rilpivirine (RPV), efavirenz (EFV), and nevirapine (NVP) in an in vitro single-cycle phenotypic assay. The amino acid substitutions E138Q/R, Y181I/V, and M230L conferred high-level resistance to ETR, while K101P and Y181I/V conferred high-level resistance to RPV. Y181C, a major NNRTI resistance-associated amino acid substitution, caused decreased susceptibility to ETR and, to a lesser extent, RPV when combined with other mutations. These included N348I and T369I, amino acid changes in the connection domain that are not generally assessed during resistance testing. However, the prevalence of these genotypes among subtype C sequences was, in most cases, <1%. The more common EFV/NVP resistance-associated substitutions, such as K103N, V106M, and G190A, had no major impact on ETR or RPV susceptibility. The low-level resistance to RPV and ETR conferred by E138K was not significantly enhanced in the presence of M184V/I, unlike for EFV and NVP. Among patient samples, 97% were resistant to EFV and/or NVP, while only 24% and 16% were resistant to ETR and RPV, respectively. Overall, only a few, relatively rare NNRTI resistance-associated amino acid substitutions caused resistance to ETR and/or RPV in an HIV-1 subtype C background, suggesting that these newer NNRTIs would be effective in NVP/EFV-experienced HIV-1 subtype C-infected patients.

Impact of the N348I Mutation in HIV-1 Reverse Transcriptase on Nonnucleoside Reverse Transcriptase Inhibitor Resistance in Non-Subtype B HIV-1

ABSTRACT We investigated the effect of N348I alone and with M184V on nonnucleoside reverse transcriptase inhibitor (NNRTI) drug susceptibility and replicative capacity in B and non-B HIV-1 isolates. N348I reduced the susceptibility to all NNRTI drugs across subtypes. The replication capacity of all viruses in a variety of cell lines was impaired by N348I. Interestingly, the N348I and M184V double mutation compensated for the reduced NNRTI drug susceptibility observed in the N348I single mutant and marginally improved viral replicative capacity.

Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial.

Background Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. Methods Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. Results We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. Conclusions Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic determinants of protease inhibitor failure in those who fail without traditional resistance mutations whilst PI use is being scaled up globally.

Rates of HIV-1 superinfection and primary HIV-1 infection are similar in female sex workers in Uganda

Objective:To determine and compare the rates of HIV superinfection and primary HIV infection in high-risk female sex workers (FSWs) in Kampala, Uganda. Design:A retrospective analysis of individuals who participated in a clinical cohort study among high-risk FSWs in Kampala, Uganda. Methods:Plasma samples from HIV-infected FSWs in Kampala, Uganda were examined with next-generation sequencing of the p24 and gp41HIV genomic regions for the occurrence of superinfection. Primary HIV incidence was determined from initially HIV-uninfected FSWs from the same cohort, and incidence rate ratios were compared. Results:The rate of superinfection in these women (7/85; 3.4/100 person-years) was not significantly different from the rate of primary infection in the same population (3.7/100 person-years; incidence rate ratio = 0.91, P = 0.42). Seven women also entered the study dual-infected (16.5% either dual or superinfected). The women with any presence of dual infection were more likely to report sex work as their only source of income (P = 0.05), and trended to be older and more likely to be widowed (P = 0.07). Conclusions:In this cohort of FSWs, HIV superinfection occurred at a high rate and was similar to that of primary HIV infection. These results differ from a similar study of high-risk female bar workers in Kenya that found the rate of superinfection to be significantly lower than the rate of primary HIV infection.

Analysis of the history and spread of HIV-1 in Uganda using phylodynamics

HIV prevalence has decreased in Uganda since the 1990s, but remains substantial within high-risk groups. Here, we reconstruct the history and spread of HIV subtypes A1 and D in Uganda and explore the transmission dynamics in high-risk populations. We analysed HIV pol sequences from female sex workers in Kampala (nu200a=u200a42), Lake Victoria fisher-folk (nu200a=u200a46) and a rural clinical cohort (nu200a=u200a74), together with publicly available sequences from adjacent regions in Uganda (nu200a=u200a412) and newly generated sequences from samples taken in Kampala in 1986 (nu200a=u200a12). Of the sequences from the three Ugandan populations, 60 (37.1u200a%) were classified as subtype D, 54 (33.3u200a%) as subtype A1, 31 (19.1u200a%) as A1/D recombinants, six (3.7u200a%) as subtype C, one (0.6u200a%) as subtype G and 10 (6.2u200a%) as other recombinants. Among the A1/D recombinants we identified a new candidate circulating recombinant form. Phylodynamic and phylogeographic analyses using BEAST indicated that the Ugandan epidemics originated in 1960 (1950–1968) for subtype A1 and 1973 (1970–1977) for D, in rural south-western Uganda with subsequent spread to Kampala. They also showed extensive interconnection with adjacent countries. The sequence analysis shows both epidemics grew exponentially during the 1970s–1980s and decreased from 1992, which agrees with HIV prevalence reports in Uganda. Inclusion of sequences from the 1980s indicated the origin of both epidemics was more recent than expected and substantially narrowed the confidence intervals in comparison to previous estimates. We identified three transmission clusters and ten pairs, none of them including patients from different populations, suggesting active transmission within a structured transmission network.

The evolution of HIV-1 reverse transcriptase in route to acquisition of Q151M multi-drug resistance is complex and involves mutations in multiple domains

BackgroundThe Q151M multi-drug resistance (MDR) pathway in HIV-1 reverse transcriptase (RT) confers reduced susceptibility to all nucleoside reverse transcriptase inhibitors (NRTIs) excluding tenofovir (TDF). This pathway emerges after long term failure of therapy, and is increasingly observed in the resource poor world, where antiretroviral therapy is rarely accompanied by intensive virological monitoring. In this study we examined the genotypic, phenotypic and fitness correlates associated with the development of Q151M MDR in the absence of viral load monitoring.ResultsSingle-genome sequencing (SGS) of full-length RT was carried out on sequential samples from an HIV-infected individual enrolled in ART rollout. The emergence of Q151M MDR occurred in the order A62V, V75I, and finally Q151M on the same genome at 4, 17 and 37 months after initiation of therapy, respectively. This was accompanied by a parallel cumulative acquisition of mutations at 20 other codon positions; seven of which were located in the connection subdomain. We established that fourteen of these mutations are also observed in Q151M-containing sequences submitted to the Stanford University HIV database. Phenotypic drug susceptibility testing demonstrated that the Q151M-containing RT had reduced susceptibility to all NRTIs except for TDF. RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility. However, the virus expressing patient-derived Q151M RT at 37 months demonstrated ~44% replicative capacity of that at 4 months. This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain.ConclusionsWe demonstrate a complex interplay between drug susceptibility and replicative fitness in the acquisition Q151M MDR with serious implications for second-line regimen options. The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.

Low Drug Resistance Levels Among Drug-Naive Individuals with Recent HIV Type 1 Infection in a Rural Clinical Cohort in Southwestern Uganda

To investigate the prevalence of transmitted drug resistance (TDR) among individuals with recent HIV-1 infection between February 2004 and January 2010 in a rural clinical cohort, samples from 72 participants were analyzed. Results from the 72 participants showed no protease inhibitor and nucleoside reverse transcriptase inhibitor-associated mutations. One participant (1.4%, 95% CI: 0.04-7.5%) had two nonnucleoside reverse transcriptase inhibitor mutations (G190E and P225H). HIV-1 subtype frequencies were A 22 (30.6%), D 38 (52.8%), and C 1 (1.4%); 11 (15.3%) were A/D unique recombinant forms. Seven years after the scale up of antiretroviral therapy (ART) in a rural clinical cohort in Uganda, the prevalence of TDR among recently HIV-1-infected individuals was low at 1.4%. Since our findings from an HIV study cohort may not be generalizable to the general population, routine TDR surveys in specific populations may be necessary to inform policy on the magnitude and prevention strategies of TDR.