Drug-associated changes in amino acid residues in Gag p2, p7NC, and p6Gag/p6Pol in human immunodeficiency virus type 1 (HIV-1) display a dominant effect on replicative fitness and drug response

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...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Parry¹,

Kohli²,

Boinett³

et al. 2009

“…The p6* transframe domain may be the most variable region in the HIV-1 Gag-Pol precursor, displaying a remarkable degree of naturally occurring and partly drug-associated polymorphisms, including insertions and deletions (2,4,15,16,35,38). The so-far-largest natural deletion of 13 amino acids was found in the p6*/p6 gag region of the Chinese isolate HIV-1 CRF07_BC and was reported to have no significant effect on viral fitness (35).…”

Section: Discussionmentioning

confidence: 99%

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

Leiherer

Ludwig

Wagner

2009

Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6 gag sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3 end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6 gag reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.

“…Twelve major protease inhibitor (PI) resistance-associated mutations have been reported in the HIV Drug Resistance Database at Stanford University (http://hivdb.stanford.edu/), and some of these reduce viral replication capacity (61)(62)(63)(64)(65)(66)(67). To address the temporal accumulation of PI resistance mutations as a potential confounder, HIV-1 protease sequences were examined for 140 subjects for whom chimeric viruses were generated (for the remaining 15 patients, PCR or sequencing was unsuccessful).…”

Section: Change In Hiv-1 Replication Capacity Over the Epidemicmentioning

confidence: 99%

Significant Reductions in Gag-Protease-Mediated HIV-1 Replication Capacity during the Course of the Epidemic in Japan

Nomura

Hosoya

Brumme

et al. 2013

f Human immunodeficiency virus type 1 (HIV-1) evolves rapidly in response to host immune selection pressures. As a result, the functional properties of HIV-1 isolates from earlier in the epidemic may differ from those of isolates from later stages. However, few studies have investigated alterations in viral replication capacity (RC) over the epidemic. In the present study, we compare Gag-Protease-associated RC between early and late isolates in Japan (1994 to 2009). HIV-1 subtype B sequences from 156 antiretroviral-naïve Japanese with chronic asymptomatic infection were used to construct a chimeric NL4-3 strain encoding plasmaderived gag-protease. Viral replication capacity was examined by infecting a long terminal repeat-driven green fluorescent protein-reporter T cell line. We observed a reduction in the RC of chimeric NL4-3 over the epidemic, which remained significant after adjusting for the CD4 ؉ T cell count and plasma virus load. The same outcome was seen when limiting the analysis to a single large cluster of related sequences, indicating that our results are not due to shifts in the molecular epidemiology of the epidemic in Japan. Moreover, the change in RC was independent of genetic distance between patient-derived sequences and wildtype NL4-3, thus ruling out potential temporal bias due to genetic similarity between patient and historic viral backbone sequences. Collectively, these data indicate that Gag-Protease-associated HIV-1 replication capacity has decreased over the epidemic in Japan. Larger studies from multiple geographical regions will be required to confirm this phenomenon.