Molecular Mechanisms of Retroviral Gag-RNA interactions in Virus Assembly

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Abstract Retroviruses are positive-sense, single-stranded RNA viruses that cause cancers and severe immunodeficiency diseases in animals and humans, including human immunodeficiency virus. Gag, the major structural protein of retroviruses, orchestrates the assembly of virus particles that bud from the plasma membrane of infected cells. To initiate particle assembly, Gag selectively binds unspliced viral RNA as the source of genomic RNA in virions. This proposal focuses on the mechanism by which Gag selects genomic RNA, addressing fundamental, unanswered questions in the field: (i) where in the cell does the initial contact between Gag and unspliced viral RNA occur; (ii) how does Gag selectively recruit unspliced viral RNA for packaging when it comprises only ~1% of the total RNA in an infected cell; and (iii) what are the properties of Gag-viral RNA complexes that promote transport through the cell to the plasma membrane for particle release? Because virus particles bud from the plasma membrane, it was originally thought that initial Gag- genomic RNA interactions occurred in the cytoplasm. Our laboratory discovered that RSV Gag undergoes nuclear trafficking, which is required for efficient genomic viral RNA packaging. This finding raised the possibility that Gag binds genomic RNA in the nucleus, which challenges the dogma for how retroviruses package their genomes. Our imaging and biophysical studies have revealed that the RSV Gag protein forms discrete foci in the nucleus, cytoplasm, and at the plasma membrane that have properties of biomolecular condensates (BMCs), which have been shown to be important in regulating cell biology processes and virus-host interactions. We have observed that the Gag nuclear foci colocalize with unspliced viral RNA, suggesting that RSV Gag initially binds genomic RNA in the nucleus. In Aim 2 of this funded project, we are using biophysical approaches to examine whether Gag-genomic RNA complexes exhibit properties of BMCs and undergo liquid-liquid phase separation. In addition, we are testing the hypothesis that Gag forms BMCs with cellular transcription factors including Mediators and RNA polymerase II to facilitate genomic RNA packaging and virion assembly. The equipment being requested in this administrative supplement, the SpectraMax® Paradigm® Multi-Mode Microplate reader with accompanying cartridges, will be used to perform the biophysical experiments described in Aim 2. This equipment will allow us to perform state-of-the-art, high throughput, quantitative assays to accomplish the goals of our approved experimental plan and publish the highest impact manuscripts.