Characterization of hepatocyte cell entry factors which govern flavivirus entry

Flaviviruses are a genus of positive-sense, single-stranded ribonucleic acid (RNA) viruses. This genus includes viruses which have caused significant public health and economic concerns worldwide, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and yellow fever virus (YFV). They are spread by arthropod hosts, mosquitos and ticks in particular, and generally cause illnesses that result in symptoms which range from mild influenza-like infection to hemorrhagic fever and encephalitis (Laureti et al., 2018). Many members of the flavivirus genus have re-emerged into the spotlight due to urban growth and increasingly hospitable environments for mosquitos due to climate change. Though effective vaccines are available against a few flaviviruses, there are still no effective vaccines available against DENV, ZIKV or WNV. Additionally, there are currently no antiviral therapeutics available for flavivirus infection; management of infection is generally supportive in nature. These challenges have caused flaviviruses to be of considerable interest to the infectious diseases research community (Collins & Metz, 2017). Virus entry into a host cell is a critical step of the virus life cycle. However, the mechanism that flaviviruses employ to enter host cells is still poorly understood. Identifying the viral and cellular factors which mediate this process could certainly allow for new inroads in the development of effective flavivirus therapeutics. Using YFV as a model flavivirus, this thesis project aims to characterize the role of specific cellular factors which govern flavivirus entry. Recent genetic screens from the Douam Lab have identified a cytoskeletal protein of interest as a potential candidate protein that regulates the YFV life cycle. As such, we hypothesized that this cytoskeletal protein is a mediator of flavivirus entry into host cells. For the first portion of our study, we sought to determine if chemical inhibition of this cytoskeletal protein could reduce the susceptibility of hepatoma cell lines to flavivirus infection, using the live attenuated yellow fever virus vaccine strain 17D (YFV-17D) as a prototypical flavivirus. We subsequently found that chemical inhibition of the activation of this cytoskeletal protein restricted YFV-17D infection of hepatoma cell lines in a dose dependent manner. In addition, we confirmed that the inhibition of infection was not due to cytotoxic effects imposed by our chemical inhibitor. For the second portion of our study, we sought to determine if this cytoskeletal protein specifically regulates YFV-17D entry. To assess this, we developed a novel YFV-17D reporter system, ΔNS1-17D, which is capable of entering cells, but is unable to produce viral particles de novo. As a whole, our study provides evidence that this cytoskeletal protein plays a significant role in regulating flavivirus infection. Future experiments will employ our new ΔNS1-17D reporter system to provide conclusive evidence that this cytoskeletal protein specifically promotes YFV-17D entry into host cells, and intend to identify the cell surface molecules bound to this cytoskeletal protein which mediate this process. By developing the foundation of a larger body of work which aims to identify host cell factors that regulate flavivirus entry, our findings could contribute to the development of innovative therapeutics against flaviviruses.2023-11-18T00:00:00