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Unpaired Guanosines in the 5′ Untranslated Region of HIV-1 RNA Act Synergistically To Mediate Genome Packaging

HIV-1 must package its RNA genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5′ untranslated region play important roles in genome packaging. Although mutating one or two NC-binding sites caused only mild defects in packaging, mutating multiple sites resulted in severe defects in genome encapsidation, indicating that unpaired guanosines act synergistically to promote packaging. Our results suggest that Gag-RNA interactions occur at multiple RNA sites during genome packaging; furthermore, there are functionally redundant binding sites in viral RNA. ABSTRACT The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion genome during virus assembly. Currently, the precise mechanism that mediates the genome selection is not understood. Previous studies have identified several sites in the 5′ untranslated region (5′ UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA genome packaging has not been fully investigated. In this report, we examined the roles of single-stranded exposed guanosines at NC binding sites in RNA genome packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA packaging efficiencies were determined by comparing their prevalences in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA packaging; of the sites tested, those located within stem-loop 1 of the 5′ UTR had the most significant effects. These sites were previously reported as the primary NC binding sites by using a chemical probe reverse-footprinting assay and as the major Gag binding sites by using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in <2-fold defects in packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA packaging, suggesting redundancy in Gag-RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 genome encapsidation. IMPORTANCE HIV-1 must package its RNA genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5′ untranslated region play important roles in genome packaging. Although mutating one or two NC-binding sites caused only mild defects in packaging, mutating multiple sites resulted in severe defects in genome encapsidation, indicating that unpaired guanosines act synergistically to promote packaging. Our results suggest that Gag-RNA interactions occur at multiple RNA sites during genome packaging; furthermore, there are functionally redundant binding sites in viral RNA.