ALG-2 interacting protein X (ALIX) links HIV-1 Gag to the components of ESCRT-III. HIV-1 engages the ALIX via its nucleocapsid and LYPXnL motif in p6. Overexpression of ALIX corrects the release defect of PTAP deleted HIV-1 via LYPXnL/ALIX pathway. However, HIV-1 subtype C lacks the LYPXnL motif and hence cannot employ LYPXnL/ALIX mechanism. Though the preferential occurrences of PYXE insertion in HIV-1 C p6 is predicted to restore the ALIX binding site there is no functional proof to support these observations. In this study we show that HIV-1 construct with subtype C p6 having PTAP deletion and PYRE insertion (pNL-INp6ΔPTAP/PYRE) could respond to ALIX overexpression. Notably, conserved Phenyl alanine residue (F676) in ALIX was critical for ALIX mediated release of pNL-INp6ΔPTAP/PYRE implying the critical role of this hydrophobic patch in ALIX recruitment. In addition, we show that Nedd4-1 could also correct the release defect of pNL-INp6ΔPTAP/PYRE. Moreover, Nedd4-1 was more robust compared to ALIX in its ability to stimulate the release of pNL-INp6ΔPTAP/PYRE. Replication kinetic data highlights the positive effect of PYRE insertion on virus replication. In summary, our data reveals the functional role of PYRE insertion towards the cooperative mechanism of ALIX/Nedd4-1 in virus release in the absence of PTAP/Tsg101 pathway.
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