Initiation of HIV-1 Gag lattice assembly is required for cytoplasmic recognition of the viral genome packaging signal

The encapsidation of HIV-1 genomic RNA into virions is enabled by the binding of the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein to the structured viral RNA packaging signal ({Psi}) at the 5 end of the viral genome. However, the subcellular location and oligomeric status of Gag during the initial Gag-{Psi} encounter remains uncertain. Domains other than NC, such as capsid (CA), may therefore indirectly affect RNA recognition. To investigate the contribution of Gag domains to {Psi} recognition in a cellular environment, we performed protein-protein crosslinking and protein-RNA crosslinking immunoprecipitation coupled with sequencing (CLIP-seq) experiments. We demonstrate that NC alone does not bind specifically to {Psi} in living cells, whereas full-length Gag and a CANC subdomain bind to {Psi} with high specificity. Perturbation of the {Psi} RNA structure or NC zinc fingers affected CANC:{Psi} binding specificity. Notably, CANC variants with substitutions that disrupt CA:CA dimer, trimer or hexamer interfaces in the immature Gag lattice also affected RNA binding, and mutants that were unable to assemble a nascent Gag lattice completely lost the ability to specific bind to {Psi}. Artificially multimerized NC domains did not specifically bind {Psi}. CA variants with substitutions in inositol phosphate coordinating residues that prevent CA hexamerization were also deficient in {Psi} binding and second-site revertant mutants that restored CA assembly also restored specific binding to {Psi}. Overall, these data indicate that the correct assembly of a nascent immature CA lattice is required for the specific interaction between Gag and {Psi} in cells.