Three-dimensional human epithelial cultures as a model for evaluation of flavivirus-host interactions driving infection in the skin

PROJECT SUMMARY Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) all cause severe human disease as a result of their widespread mosquito-borne transmission. While these flaviviruses share a common primary site of infection in the dermis, they disseminate to varying secondary sites of infection with highly divergent clinical outcomes. We propose that discrepancies in early virus-host interactions among ZIKV, DENV, and WNV are responsible for these differences in disease; however, there is a gap in our knowledge of the molecular mechanisms responsible. Elucidation of distinct virus-host interactions occurring at the initial infection site is thus key to understanding how these viruses establish productive infections in the skin and elsewhere. The proposed research will establish organotypic epithelial cultures as a genetically tractable and immunocompetent 3D human skin model of flavivirus infection, to be used for virus-host interaction studies. We will expand on the current model commonly used to study DNA viruses, typically comprised solely of human fibroblasts and keratinocytes, with the addition of human skin-resident dendritic cells and macrophages, to accurately recreate natural flavivirus infection in the skin. Additionally, we will incorporate mosquito saliva, which includes immunomodulatory and anti-inflammatory proteins, into the virus inoculum to further recapitulate the molecular events occurring at the primary site of flavivirus infection. We will determine individual infection conditions for ZIKV, DENV, and WNV, and evaluate cell tropism and host responses with each virus. Once established, we will employ this 3D human skin model to evaluate the role of the host ribonuclease L (RNase L) protein during ZIKV infection in the skin, which will validate this system for flavivirus-host interaction studies. While antiviral RNase L activity is well described, we have recently discovered proviral RNase L activity during ZIKV infection. In contrast, we observed canonical antiviral RNase L activity during DENV and WNV infections. These studies were performed in 2D monolayer culture systems with immortalized cell lines, therefore we will use our 3D skin model containing primary cells to test the hypothesis that RNase L plays a role in ZIKV cell tropism and spread in the skin in a more relevant system. We will use CRISPR-Cas9 gene editing to delete RNase L from the different skin cells comprising epithelial cultures, and subsequently generate RNase L-deficient skin cultures for infections with ZIKV, DENV, or WNV. We will assess effects of RNase L deletion on infection and spread of the different flaviviruses in the skin, as well as how host responses are impacted. The proposed studies will characterize the role of RNase L as an important host factor repurposed by ZIKV during infection in the skin. Furthermore, this project will establish organotypic epithelial cultures as an exciting new human model amenable to gene editing which will facilitate inquiries of arbovirus-host interactions and host responses at the common primary infection site of the skin.