Abstract
During retrovirus assembly, Gag packages unspliced viral RNA as the virion genome. Genome packaging is usually specific with occasional exceptions of cross-packaging RNA from distantly related retroviruses. For example, HIV-1 Gag can efficiently package HIV-2 RNA. To better understand how HIV-1 Gag selects packaging substrates, we defined elements in the HIV-2 5' untranslated region (UTR) that are important for this process. Although sharing little homology, both HIV-1 and HIV-2 5' UTRs have unpaired guanosines essential for packaging by their own Gag. Simultaneously substituting guanosines of nine sites in the HIV-2 5' UTR caused severe defects in HIV-1 Gag-mediated packaging. Two of the nine sites are particularly important, mutating each one impaired HIV-1 Gag-mediated packaging, whereas the other sites required mutations in multiple sites to produce similar effects. Additionally, we identified one site that impacts HIV-1 Gag but is dispensable for HIV-2 Gag selective packaging. Furthermore, combining mutations has an additive effect on packaging defects for HIV-1 Gag, in contrast to the previously reported synergistic effects for HIV-2 Gag. Our study demonstrates that Gag proteins from two different retroviruses recognize and use mostly the same set of cis-acting elements to mediate RNA packaging and provide the mechanistic basis for genome cross-packaging.