Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain

Abstract

HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-AlaPro (PTAP), located in the p6 region of Gag (p6 Gag ), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6Gag significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6Gag confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy. IMPORTANCEResistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

HIV-1 subtype C with PYxE insertion has enhanced binding of Gag-p6 to host cell protein ALIX and increased replication fitness

“…Therefore, this domain appears to be necessary for viral budding. Although one report shows that PTAP duplication only increases HIV-1 replication in the presence of a PI (26), others and we observed increased viral growth when HIV-1 contained a PTAP duplication (25). Although deletion of the PTAP motif has not been identified in clinical strains so far, artificial deletion or mutation of this PTAP motif severely abrogates release of HIV-1 from infected cells (11, 32, 33).…”

“…We also included two HIV-1C strains with a PTAP duplication (PT02 and PT03) as it has been shown to increase the viral fitness and affect the drug sensitivity (Fig. 5a) (25, 26). Then, these sequences were cloned into the genome of a reference virus (NL4-3, HIV-1B wild type) replacing its gag-pol sequence.…”

Increased replication capacity following evolution of PYxE insertion in Gag‐p6 is associated with enhanced virulence in HIV‐1 subtype C from East Africa

“…The relatively low number of failures did not allow us to observe any association with Gag p6 genotype and viral failure. A recent study reported that PTAP-duplications enhance the resistance to several protease inhibitors (PIs) [Martins et al, 2015]. However, in our drug sensitivity assay including all available PIs, no difference was observed in EC 50 fold change between either PTAPduplicated HIV-1B or HIV-1C viruses (Supplementary File S1).…”

Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner

“…First, we altered the N-terminus as the suspected primary binding site for IDE [48] by duplication of the N-terminal PTAP-motif, a mutation that is frequently observed in clinical HIV-isolates as it augments virus maturation under antiviral treatment [49,50]. To mimic these natural N-terminal alterations, we generated a p6 mutant containing two PTAPPA domains (further referred to as 2x, Fig 8B) and tested its stability by in vitro degradation assays using S10 and rIDE combined with synthetic or virally-produced substrates of the 2x mutant version.…”