Mutations in HIV-1gagandpolCompensate for the Loss of Viral Fitness Caused by a Highly Mutated Protease

Abstract

f During the last few decades, the treatment of HIV-infected patients by highly active antiretroviral therapy, including protease inhibitors (PIs), has become standard. Here, we present results of analysis of a patient-derived, multiresistant HIV-1 CRF02_AG recombinant strain with a highly mutated protease (PR) coding sequence, where up to 19 coding mutations have accumulated in the PR. The results of biochemical analysis in vitro showed that the patient-derived PR is highly resistant to most of the currently used PIs and that it also exhibits very poor catalytic activity. Determination of the crystal structure revealed prominent changes in the flap elbow region and S1/S1= active site subsites. While viral loads in the patient were found to be high, the insertion of the patient-derived PR into a HIV-1 subtype B backbone resulted in reduction of infectivity by 3 orders of magnitude. Fitness compensation was not achieved by elevated polymerase (Pol) expression, but the introduction of patient-derived gag and pol sequences in a CRF02_AG backbone rescued viral infectivity to near wild-type (wt) levels. The mutations that accumulated in the vicinity of the processing sites spanning the p2/NC, NC/p1, and p6pol/PR proteins lead to much more efficient hydrolysis of corresponding peptides by patient-derived PR in comparison to the wt enzyme. This indicates a very efficient coevolution of enzyme and substrate maintaining high viral loads in vivo under constant drug pressure.

Evolutionary consequences of drug resistance: shared principles across diverse targets and organisms

“…An interesting example is that of drug resistance in HIV that is caused by multiple mutations in the protease that reduce fitness: the fitness consequences can be rescued by mutations in the vicinity of the viral Gag proteolytic cleavage sites, leading to improved processing of Gag by the highly mutated protease 49 .…”

“…Different proteins are shown as light blue and light green colours. d | A bypass mechanism whereby, for example, an additional regulatory factor compensates for the reduced functionality of the mutant protein 19,49 .…”

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

“…Structural modeling suggests that the mutations in the cleavage site act to improve interactions between the substrate and the mutated protease and increase the efficiency of cleavage. Hence, mutations in the Gag and Gag-Pol substrates can compensate for the lower catalytic efficiency of resistant protease mutants [18,19]. This mechanism of resistance can be difficult to detect as the protease genotype from the patient would not necessarily match the expected PI-resistance profile [20].…”

Novel Dengue Virus NS2B/NS3 Protease Inhibitors

“…Viruses were produced by transfection of HEK 293T cells with proviral plasmid pNL4-3 encoding infectious HIV-1 subtype B as described before (34). Viral titers were determined on TZM-bl indicator cells (CD4 ϩ CCR5 ϩ CXCR4 ϩ HeLa cells) in singlecycle infections by serial dilutions of the cell culture supernatants as previously described (33,34). Briefly, cell culture supernatants were collected, centrifuged (1,500 rpm, 5 min) to remove transfected HEK 293T cells, and titrated in serial dilutions on TZM-bl cells (96-well plate, 2 ϫ 10 4 cells per well).…”