Mechanistic studies of genomic RNA dimerization in an oncoretrovirus

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PROJECT ABSTRACT Retroviruses are single-stranded RNA viruses that cause cancers and immunodeficiency diseases in humans and animals. Our laboratory studies Rous sarcoma virus (RSV), an avian retrovirus that causes solid tumors in domesticated fowl, as a model for dissecting the molecular underpinnings of retroviral assembly. Our discovery that the RSV structural protein Gag transiently travels through the nucleus in a Crm1-dependent fashion challenged the traditional view of retroviral assembly. Treatment with a Crm1 inhibitor or mutation of the nuclear export signal (NES) effectively traps RSV Gag in the nucleus, allowing us to further study the role of Gag in the nucleus. Recent studies have revealed additional retroviral Gag proteins that undergo nuclear localization, suggesting there may be common functions of Gag within the nucleus. Retroviruses are unique in that they package two copies of their genome as non-covalently linked genomic RNA dimers. Genome dimerization is facilitated by cis-acting sequences located in the 5’UTR of the viral RNA. Dimerization of the genome is conserved in orthoretroviruses and is required for viral replication. Despite its critical importance in replication, the mechanisms underlying genome dimerization within infected cells remains poorly understood. RSV affords a unique opportunity to investigate genome dimerization using our extensive toolbox of genetic, biochemical and imaging methods because we can readily manipulate the subcellular localization of Gag with our well-characterized collection of viral mutants. In recent work, we have developed methods to visualize fluorophore-tagged RSV Gag proteins co-localized with viral genomic RNA in the cell. Our experiments suggest nuclear trafficking of Gag is required for the efficient packaging of retroviral genomic RNA. This proposal thus aims to understand the mechanism governing genome dimerization in RSV, and we will test the hypothesis that nuclear trafficking of Gag plays a role in facilitating genome dimerization. In these studies, we will utilize the MS2 and Bgl RNA labeling system to visualize two distinct viral RNA populations to examine genome dimerization. This approach will allow us to determine the subcellular location of dimerization; whether there is preferential formation of heterodimers, which contain two genetically distinct viral genomes, or homodimers, composed of two identical viral genomes; and whether dimerization occurs in a co-transcriptional manner. We will also examine the role of nuclear Gag in genome dimerization and determine whether RSV Gag initially binds monomers or dimers. Studies that express the viral RNA and Gag in trans will investigate whether Gag facilitates genome dimerization within different compartments of the cell. The findings from this proposal will greatly contribute to the understanding of this critical step in the replication cycle and may provide support for future anti-retroviral therapeutics. Overall, this program is designed to provide a comprehensive experience consisting of opportunities in laboratory-based research, scientific career development, educational seminars, and mentoring to advance physician-scientist training.