SRA

The HIV-1 surface glycoprotein Env binds target receptors to mediate fusion of the viral and host cell membranes during infection. Env is incorporated with very low density into virions, and in some cell types, regions within the long cytosolic C-terminal tail may mediate direct or indirect associations with Gag during virus assembly and budding. Here, the mutational landscape is determined across the transmembrane and proximal cytosolic domains of Env (001428 isolate from clade C) interacting with the MA domain of Gag at cellular membranes. No evidence is found in a derivative line of HEK293 cells for specific motifs that mediate Env/MA associations. Overall design: A single site-saturation mutagenesis (SSM) library was constructed in the Env protein from strain 001428, focused on residues N677-L753 (HXB2 reference numbering). Env was truncated at residue L753 for enhanced surface expression, and fused via a GGSG linker to the C-terminal half (VC) of split Venus. The 001428(753)-VC Env library was transfected into Expi293F cells stably expressing MA fused to the N-terminal half (VN) of split Venus. When 001428(753)-VC and MA-VN are in close physical proximity, the respective halves of Venus can fold into a functional fluorophore. This method for investigating protein interactions within living cells is known as bimolecular fluorescence complementation (BiFC). The transfected cells were screened by fluorescence-activated cell sorting (FACS) for Env expression based on staining with the broadly neutralizing antibody PG16, and for either high or low BiFC signal. Transcripts in the sorted cell populations were deep sequenced, and the frequencies of all single amino acid substitutions were compared to the naive plasmid library. Sorting experiments were independently replicated.