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

Human immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Through in silico predictions and in vitro experiments, we showed that HIV-1C Gag has an increased binding to ALIX when PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derived gag-pol sequences from HIV-1C PYxEi viruses and inserted them in a reference HIV-1. Viral growth was increased, and the sensitivity to protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion could indicate the clinical importance of these viral variants. Importance Genomic differences within HIV-1 subtypes is associated with a varying degree of viral spread, disease progression, and clinical outcome. Viral budding is essential in the HIV-1 life cycle and mainly mediated through the interaction of Gag with host proteins. Two motifs within Gag-p6 mediate binding of host cell proteins and facilitate budding. HIV-1 subtype C (HIV-1C) has a natural deletion of one of these two motifs resulting in an inability to bind to host cell protein ALIX. Previously, we have identified a tetrapeptide (PYxE) insertion at this deleted motif site in a subset of HIV-1C patients. Here, we report the incidence of PYxE insertions in three different HIV-1C cohorts, and the insertion restores the binding of Gag to ALIX. It also increases viral growth even in the presence of antiretroviral drugs lopinavir and tenofovir alafenamide. Hence, PYxE insertion in HIV-1C might be biologically relevant for viruses and clinically significant among patients.