HIV-1 RNA genomes initiate host protein packaging in the cytosol independently of Gag capsid proteins

HIV-1 RNA genomes interact with diverse RNA binding proteins (RBPs) in the cytoplasm including antiviral factor APOBEC3G (A3G) that, in the absence of viral Vif proteins, is packaged into virions. Where and when HIV-1-A3G interactions are initiated for packaging inside the cell is unknown, and the relative contributions of genome vs. Gag capsid proteins to this process remains controversial. Here we visualized A3G responses to HIV-1 infection over an entire replication cycle using long-term (up to 72 h) live single cell imaging. We show that Vif-deficient HIV-1 dramatically shifts A3G and a second RNA surveillance factor, MOV10, from the cytoplasm to virus particle assembly sites with little to no net discernible effects on general markers of cytoplasmic processing bodies (DCP1A), stress granules (TIA1), or a marker of the nonsense-mediated decay machinery (UPF1). Using a new live cell RNA-protein interaction assay based on RNA tethering (the in-cell RNA-protein interaction protocol, or IC-IP), we provide evidence that A3G- and MOV10- genome interactions are selective, strong, occur in presence or absence of Gag, and are initiated in the cytosol soon if not immediately after genome nuclear export. Finally, although Gag is sufficient to package A3G into virions even in the absence of genomes, single virion imaging indicates that selective A3G-genome interactions promote much more consistent per virion delivery of A3G to assembly sites. Collectively, these studies suggest a paradigm for early, strong, and persistent cytosolic detection of select HIV-1 RNA signatures by A3G, MOV10 and other host RBPs that are enriched in virions.