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.The development of antiretroviral drugs and their use in highly active antiretroviral therapy have led to the ability to effectively control human immunodeficiency virus (HIV) replication in infected patients. The emergence of resistance to these drugs, while reduced when the drugs are used in combination, remains a problem. A recent study has estimated that by 10 years of treatment, nearly 10% of patients will have experienced resistance to the three main classes of drugs that form the basis of highly active antiretroviral therapy. Further, the risk of death within 5 years after extensive triple-class failure is 10% (31). Resistance develops due to several factors, including the high error rate of reverse transcriptase (RT), viral recombination, high viral turnover, and suboptimal drug levels in patients. Mutations emerging in viruses can lead to cross-class resistance.Resistance to protease inhibitors (PI) occurs by the stepwise accumulation of mutations in protease itself, many of which lead to a reduced replication capacity (RC) of the virus (5-7, 22, 23, 37). Primary, or major, mutations mostly lead to a reduced affinity for the inhibitor through alterations in the enzyme active site. Further secondary, or minor, mutations also arise in protease that have little effect on inhibitor binding and therefore do not convey res...

Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients

“…Gag and PR function as a single unit with enzyme and substrate coevolving to optimize viral replication. 32 Mutations at the Gag CS A431V, L449P, and P453L have been shown to contribute to PI resistance.…”

Novel Two-Round Phenotypic Assay for Protease Inhibitor Susceptibility Testing of Recombinant and Primary HIV-1 Isolates

“…Several algorithms are currently available for the interpretation of genotyping results; however, only mutations in the HIV-1 protease-coding region are taken into account for the inference of susceptibility to PIs. Other Gag mutations affect susceptibility to PIs, not only at cleavage sites (8,21,26) but also at noncleavage residues located at the matrix (p17) and capsid (p24) coding regions (40,41). Even so, current standard phenotypic assays do not include the patient-derived full-length Gag region in the recombinant viruses used for PI susceptibility evaluation.…”

“…Nevertheless, viral input is not generally normalized in most phenotypic assays. Here, the use of the fulllength patient-derived viral genome may help to recover viral fitness, as compensatory mutations along the Gag region restore otherwise compromised activity of viruses containing multiple amino acid substitutions associated with resistance to PIs (8,9,12,26,37,40,41,46,47,55). Nevertheless, most of the samples in these studies were B subtype, and further research is needed to better define PI resistance determinants and compensatory effects in non-B subtypes.…”

“…These viral isolates were generated from the plasma samples of a patient who had experienced successive virological failures with PI-containing antiretroviral therapy. Further studies should be performed to determine whether mutations in the 5= half region of Gag of these primary isolates (not included in the recombinant viral construction used in the Antivirogram assay) cause higher levels of resistance when full-length viruses are tested since the direct contribution of residues in HIV matrix to PI resistance and the coevolution of these residues with drug-resistant proteases have been reported (40,41).…”

“…To evaluate viral susceptibility to protease inhibitors (PIs), other noninfectious phenotypic methods have been developed to measure the in vitro activity of recombinant patient-derived HIV-1 proteases, either in cell-based settings or in cell-free experimental settings (3,18,19,27,28). However, the recently reported additional effect of Gag on restoration of viral fitness and on susceptibility to PIs is brought about mainly by mutations located at the p7/p1 and p1/p6 cleavage sites (8,9,21,26,46,55), as well as in other residues along the Gag polyprotein sequence (12,37,40,41,47). Of note, preexistent polymorphisms at p2/p7 have been associated with virological failure in a first-line PI monotherapy regimen, despite the absence of major PI resistance mutations in the protease gene (13).…”