Abstract
HIV-1 subtype C (HIV-1C) may duplicate longer amino acid stretches in the p6 Gag protein, leading to the creation of an additional Pro-Thr/Ser-Ala-Pro (PTAP) motif necessary for viral packaging. However, the biological significance of a duplication of the PTAP motif for HIV-1 replication and pathogenesis has not been experimentally validated. In a longitudinal study of two different clinical cohorts of select HIV-1 seropositive, drug-naive individuals from India, we found that 8 of 50 of these individuals harbored a mixed infection of viral strains discordant for the PTAP duplication. Conventional and next-generation sequencing of six primary viral quasispecies at multiple time points disclosed that in a mixed infection, the viral strains containing the PTAP duplication dominated the infection. The dominance of the double-PTAP viral strains over a genetically similar single-PTAP viral clone was confirmed in viral proliferation and pairwise competition assays. Of note, in the proximity ligation assay, double-PTAP Gag proteins exhibited a significantly enhanced interaction with the host protein tumor susceptibility gene 101 (Tsg101). Moreover, Tsg101 overexpression resulted in a biphasic effect on HIV-1C proliferation, an enhanced effect at low concentration and an inhibitory effect only at higher concentrations, unlike a uniformly inhibitory effect on subtype B strains. In summary, our results indicate that the duplication of the PTAP motif in the p6 Gag protein enhances the replication fitness of HIV-1C by engaging the Tsg101 host protein with a higher affinity. Our results have implications for HIV-1 pathogenesis, especially of HIV-1C.
Highlights
HIV-1 subtype C (HIV-1C) may duplicate longer amino acid stretches in the p6 Gag protein, leading to the creation of an additional Pro–Thr/Ser–Ala–Pro (PTAP) motif necessary for viral packaging
6 The abbreviations used are: LTR, long terminal repeat; ANOVA, analysis of variance; DAPI, 4,6-diamidino-2-phenylindole; NGS, next-generation sequencing; ICS, intracellular cytokine staining; PBMC, peripheral blood mononuclear cell; IFN, interferon; CFSE, carboxyfluorescein succinimidyl ester; HTA, heteroduplex tracking assay; m.o.i., multiplicity of infection; PTAP, Pro–Thr/Ser–Ala–Pro; NC, nucleocapsid; ESCRT, endosomal sorting complex required for transport; ART, anti-retroviral therapy; VT, variant type; PLA, proximity ligation assay; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; minority variants” (MV), minority variant; pol, polymerase
We demonstrated recently that in two different regions of the viral genome, HIV-1C exploits the phenomenon of sequence duplication to gain replication advantage by increasing the number of the sequence motifs of biological significance
Summary
HIV-1 subtype C (HIV-1C) may duplicate longer amino acid stretches in the p6 Gag protein, leading to the creation of an additional Pro–Thr/Ser–Ala–Pro (PTAP) motif necessary for viral packaging. Our results indicate that the duplication of the PTAP motif in the p6 Gag protein enhances the replication fitness of HIV-1C by engaging the. Sequence variations in the viral genome can influence viral biological properties that in turn may influence differences in the prevalence of the viral subtypes One such genetic variation has been observed in HIV-1 p6 Gag consisting of the amino acid sequence duplication of the Pro–Thr/Ser–Ala–Pro motif, referred to as the PTAP motif [15]. The Gag proteins collectively play an important role in viral replication by regulating viral assembly, budding, and maturation of the virions [17]. The NC by associating with SP2 and p6 recruits Alix, an important member of the host endosomal sorting complex required for transport (ESCRT), thereby regulating viral budding [22]. p6, the smallest domain of Gag, plays a major role
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