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

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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 (EC 50 ). 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 ␣-helix-4 of matrix, leading to the hypothesis that more flexibility within this region or altered matrix structure would account for our findings.Current British HIV Association (BHIVA) and other guidelines for highly active antiretroviral therapy (HAART) in the treatment of HIV and AIDS recommend first-line therapy with three active drugs: two nucleoside reverse transcriptase (RT) inhibitors and a nonnucleoside RT inhibitor. Protease inhibitors (PIs) are used with two active RT inhibitors in second-line therapy after the failure of first-line therapy (11). PIs are some of the most potent of the antiretroviral drugs in HIV clinical practice. Resistance to PIs develops by the accumulation of mutations within the protease gene that change amino acids within the enzymatic active site, reducing the binding of the inhibitor. Many of these primary PI resistance mutations have a negative effect on virus fitness or replication capacity, resulting in further secondary mutations that do not cause resistance themselves but instead increase the replication capacity of the resistant virus (2-5, 20, 22, 26). HIV protease cleaves Gag and Gag-Pol polyproteins, resulting in viral maturation after cellular release. Mutations within the Gag protein, particularly at the cleavage sites (cleavage site mutations [CSMs]) have also been associated with the recovery of replication capacity (9,10,24,31,35) as well as with PI resistance without protease mutations (27). Structural analysis showed that the A431V CSM has incr...

Section: Discussionsupporting

confidence: 81%

mentioning

confidence: 99%

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

Parry¹,

Kolli

Cane³

et al. 2011

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“…A retroviral vector-based, single-round infection system was used for in vitro phenotyping (28)(29)(30)(31)(32). For this, a 3,500-bp fragment spanning gag and part of the integrase gene (bp 737 to 4,403 of strain HXB2) of the HIV-1 subtype B p8.9NSX vector (30) was replaced with the subtype C MJ4 proviral genome (GenBank accession number AF321523.1) (31,33). NNRTI resistance-associated mutations were introduced into the p8.MJ4 plasmid using a QuikChange II XL sitedirected mutagenesis kit (Stratagene, La Jolla, CA, USA) in conjunction with mutagenesis primers synthesized by Integrated DNA Technologies (Coralville, IA, USA).…”

Section: Hiv-1 Subtype C Sequencesmentioning

confidence: 99%

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

Basson

Rhee

Parry

et al. 2015

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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. Highly active antiretroviral therapy (HAART), which comprises the concomitant use of multiple potent antiretroviral drugs, has contributed to a significant decrease in the morbidity and mortality of people infected with HIV-1 (1). The failure of HAART through the acquisition of HIV drug resistance-associated substitutions that cause a decrease in viral susceptibility is usually due to poor adherence and insufficient drug concentrations. Although more than two-thirds of the global HIV-1 infections occur in sub-Saharan African countries, where HIV-1 infections are dominated by non-B subtypes, HAART regimens have largely been developed and tested against HIV-1 subtype B isolates (2). Subtype C accounts for almost half of all global infections and dominates the epidemic in southern Africa (3). While HAART agents are effective against all subtypes (4), greatly aiding the global response to HIV infection, specific resistance mutations and disparities in drug susceptibilities can differ by subtype (5). Examples include the K65R (6) and V106M (7) resistance-associated amino acid substitutions, which develop more frequently under drug pressure in HIV-1 subtype C than subtype B.Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are a component of most first-line HAART regimens. For efavirenz (EF...

“…Since PIs inhibit the cleavage of viral proteins, this prevents the formation of mature infectious particles (17). PI resistance mutations on their own reduce viral fitness; however, amino acid substitutions at the Gag CS and non-CS that restore viral fitness in the presence of protease sub-stitutions have been identified (1,(18)(19)(20)(21)(22). Furthermore, Gag CS substitutions at positions 431, 436, and 437 have been associated with a reduction in PI susceptibility and virological failure during PI therapy in the absence of protease substitutions (23,24).…”

mentioning

confidence: 99%

Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy

Giandhari

Basson

Sutherland

et al. 2016

f Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicative in vitro assay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of the gag gene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.