A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

Abstract

BackgroundHIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors.Methods and FindingsWe have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy.ConclusionsHIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

Non-Cleavage Site Gag Mutations in Amprenavir-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) Predispose HIV-1 to Rapid Acquisition of Amprenavir Resistance but Delay Development of Resistance to Other Protease Inhibitors

“…However, a recombinant HIV-1 clone containing the protease of HIV APVp33 a n d t h e fi v e G a g n o n -c l e a v a g e s i t e m u t a t i o n s , rHIV APVp33pro 12/75/219/390/409gag , replicated moderately under the same conditions ( Fig. 2A), an observation in agreement with reports by others that some PI resistance-associated mutations compromise the catalytic activity of protease and/or alter polyprotein processing, often leading to slower viral replication (29,36,43). Since some of the five non-cleavage site mutations emerged before mutations in protease developed, we examined the effects of three sets of non-cleavage site amino acid mutations upon the emergence of APV resistance.…”

“…Of note, a number of PI resistanceassociated amino acid substitutions in the active site of protease have been identified, and such substitutions have considerable impact on the catalytic activity of protease. This impact is reflected by impaired processing of Gag precursors in mutated-protease-carrying virions and by decreased catalytic efficiency of the protease toward peptides with natural cleavage sites (7,29,31,43).…”

“…These mutations have been identified in PIresistant HIV-1 variants selected in vitro (2,5,8,29) and in HIV-1 isolated from patients with AIDS for whom chemotherapy including PIs was failing (26,40,47,48). These mutations are known to compensate for the enzymatic impairment of protease, per se, resulting from the acquisition of PI resistance-conferring mutations within the proteaseencoding region.…”

Induced Maturation of Human Immunodeficiency Virus

“…Data from mutational analyses suggest that tight temporal control of processing at individual sites is required in order to correlate RNP condensation with mature capsid assembly. A crucial importance of NC-SP2-p6 processing dynamics is also implied by the mapping of resistance-associated mutations restoring viral fitness in PI-resistant HIV-1 to this region (49). Failure to faithfully reconstitute wt processing kinetics may thus result in aberrant particle morphology.…”

Ninety-Nine Is Not Enough: Molecular Characterization of Inhibitor-Resistant Human Immunodeficiency Virus Type 1 Protease Mutants with Insertions in the Flap Region

“…In addition to primary and secondary mutations in the PR itself, amino acid changes in the cleavage sites of viral Gag polyprotein are also introduced in order to improve the ability of the mutated enzyme to bind and cleave its substrate (12,46,22). Recently, a novel mechanism of PI resistance involving primary mutations in the natural substrate leading to improved polyprotein processing without any prior mutations in the PR coding region was identified (28). Occasionally, amino acid insertions instead of substitutions are selected during antiretroviral therapy.…”