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The principal structural component of a retrovirus particle is the Gag protein. Retroviral genomic RNAs contain a 'packaging signal' ('Ψ') and are packaged in virus particles with very high selectivity. However, if no genomic RNA is present, Gag assembles into particles containing cellular mRNA molecules. The mechanism by which genomic RNA is normally selected during virus assembly is not understood. We previously reported (Comas-Garcia et al., 2017) that at physiological ionic strength, recombinant HIV-1 Gag binds with similar affinities to RNAs with or without Ψ, and proposed that genomic RNA is selectively packaged because binding to Ψ initiates particle assembly more efficiently than other RNAs. We now present data directly supporting this hypothesis. We also show that one or more short stretches of unpaired G residues are important elements of Ψ; Ψ may not be localized to a single structural element, but is probably distributed over >100 bases., Competing Interests: MC, TK, SD, DH, WH, AR No competing interests declared

Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis -acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.