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

Abstract

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.

Cytotoxic Lymphocytes Target HIV-1 Gag Through Granzyme M-Mediated Cleavage

“…It has been suggested that Gag can be in a compact conformational structure when in solution or an extended conformational structure when bound to the plasma membrane when still at the immature non-processed state, although these observations were demonstrated with Gag molecules that are lacking the P6 domain (29). Gag is predominantly localized at the plasma membrane (16,30,31), and the P6 domain plays an important role prior and during the virion assembly phase (15). Therefore, this domain is exposed to binding to other viral and host cell proteins and thus also exposed for targeting by GrM.…”

“…Therefore, this domain is exposed to binding to other viral and host cell proteins and thus also exposed for targeting by GrM. Even though only a relatively small 2 kDa C-terminal part of the P6 late domain is being cleaved off, this exposed domain is important for its interaction with host cell protein ALIX and serves as a binding domain for HIV-1 accessory protein Vpr (16,(32)(33)(34)(35)(36)(37). The GrM cleavage site is directly after the ALIX binding motif and cleavage could disturb the interaction of ALIX with Gag and thereby affecting the budding of virions from the plasma membrane (16,33).…”

“…Even though only a relatively small 2 kDa C-terminal part of the P6 late domain is being cleaved off, this exposed domain is important for its interaction with host cell protein ALIX and serves as a binding domain for HIV-1 accessory protein Vpr (16,(32)(33)(34)(35)(36)(37). The GrM cleavage site is directly after the ALIX binding motif and cleavage could disturb the interaction of ALIX with Gag and thereby affecting the budding of virions from the plasma membrane (16,33). The motif for binding Vpr to Gag, LXSLFG, is located after the GrM cleavage site and deletion of this motif completely abolishes the incorporation of Vpr into virions (32,(34)(35)(36)(37).…”

“…pCR3.1/HIV1B-Gag-mCherry (HIV-1B Gag) was a kind gift from Dr. Paul Bieniasz (The Rockefeller University, USA). pCR3.1/ HIV1C-Gag-mCherry variants with or without PYKE or PYQE tetrapeptide insertions were described previously (16). The Gag PYKEi-L483A mutant was generated by PCR-directed cloning using pCR3.1/HIV-GagPYKEi-mCherry as template.…”

“…The PYKE motif is important for the interaction of the viral Gag to host cell protein ALIX that facilitates viral budding. Furthermore, insertion of this motif correlated with enhanced viral fitness (16,17). Interestingly, HIV-1C Gag with the PYKE insertion (Gag PYKEi ) contains the GrM consensus substrate motif KEPL overlapping the PYKE motif, whereas HIV-1B and HIV-1C without the tetrapeptide insertion only contain the proline and leucine residues at this site within Gag.…”