Research Projects

Dengue virus (DENV) is a single stranded positive sense RNA virus that infects ~400 million people every year. Although the entire viral life cycle is completed in the cytoplasm, the DENV capsid protein can localize to the nucleus in infected cells. Mutations and small molecule compounds that block nuclear import of the capsid inhibit viral assembly and virus-induced apoptosis, suggesting that the capsid may play additional roles beyond viral RNA packaging and influence nuclear processes. However, the functional roles of the DENV capsid in the nucleus are not well characterized. Current studies exploring the DENV capsid interactome do not differentiate between nuclear and cytoplasmic interactions of the capsid. Recombinant DENV genomes with mutations in the capsid nuclear localization signal (NLS) are not viable, making it difficult to pinpoint its nuclear roles. In Aim 1 of the proposed study, we will establish cell lines stably expressing either the wildtype or the NLS mutant DENV capsid proteins. We will use crosslinking mass-spectrometry to identify and compare capsid-interacting proteins in these cell lines, as well as in nuclei extracted from DENV-infected cells to uncover unique, nucleus-specific interactions of the capsid. Next, we will investigate the roles of these nuclear proteins in the DENV life cycle, assessing how their expression, localization, and functions such as ribosome biogenesis, cell cycle regulation, and RNA processing are altered during infection. We expect to identify novel capsid-nuclear protein interactions that impact viral infection and pathogenesis. The DENV capsid also interacts with nuclear histones and DNA, disrupting histone oligomerization and nucleosome formation. This points to a novel epigenetic mechanism by which the capsid could regulate gene expression in infected cells. In Aim 2, we will validate interactions of the DENV capsid with nuclear DNA and histones and further investigate if it modulates chromatin accessibility and cellular gene expression by performing micrococcal nuclease assays, RNA-seq and ATAC-seq analyses in cell lines expressing the wildtype or NLS mutant capsid proteins, as well as in DENV-infected cells. We will validate the role of the capsid in mediating these changes by chromatin immune-precipitation with capsid-specific antibodies and study their role in shaping host responses to DENV infection. We expect to uncover novel pathways that are transcriptionally and epigenetically modulated by the nuclear capsid. Finally, in Aim 3, we will evaluate the effects of known pharmacological inhibitors that inhibit nuclear import of the capsid on viral infection and capsid-regulated pathways identified in Aims 1 and 2.The project will provide new insights into the role of the DENV capsid in regulating viral infection by modulating the nuclear proteome and transcriptome and identify effective inhibitors targeting the nuclear capsid that can reduce viral titers and mitigate viral pathogenicity.