Mutations in multiple domains of Gag drive the emergence of in vitro resistance to the phosphonate-containing HIV-1 protease inhibitor GS-8374

Abstract

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.

Fig 1

Structure of GS-8374.

Fig 2

Time course of the in vitro HIV-1 resistance selection in the presence of GS-8374, lopinavir (LPV), or atazanavir (ATV). Arrows indicate the time points at which viral isolates were collected for genotypic and phenotypic analysis.

Fig 3

Mutations present in the Gag-Pol transframe region of the GS-8374-resistant virus (8374-P62). (A) Amino acid changes in Gag and Pol open reading frames. PR cleavage sites are depicted in blue, and mutations in 8374-P62 are highlighted in red. The green box represents the frameshift-controlling sequence. (B) Location of the mutations in RNA hairpin that controls the Gag-Pol frameshift (g2097a and t2110c).

Fig 4

Recombinant HIV-1 variants expressing various subsets of mutations identified in the GS-8374–P62 isolate. M1 to M11 represent individual recombinant variants with the corresponding fold resistance to GS-8374 in the column on the right. HIV-1 variants with reduced susceptibility to GS-8374 are highlighted in red.

Fig 5

Proteolytic processing of Gag in recombinant mutant HIV-1 variants. Control wild-type and individual mutant viruses (designations identical to those shown in Fig. 4) were produced by transfection of proviral DNA into 293T cells in the absence or presence of GS-8374, and the products of Gag maturation were analyzed by quantitative immunoblot (LI-COR) using polyclonal antisera raised against recombinant HIV-1 MA, CA, and NC, respectively (see Fig. S1 in the supplemental material). The band intensities were quantitated using LiCor Odyssey 2.0 software. The relative amounts of mature MA, CA, and NC products are expressed as a percentage of total Gag-derived species. The data were derived from Fig. S1 in the supplemental material.