During the past 30 years, there have been several Flaviviridae threats. Among them, Hepatatis C virus (HCV) emerged in the Western hemisphere as the previously unidentified etiological agent of non-A non-B hepatitis in transfusion patients. Less than twenty-five years after the 1989 discovery of HCV, several high-efficacy direct acting antivirals (DAAs) boasting a >95% cure rate were approved for treatment of HCV infected patients. While the advent of these DAAs has revolutionized the prognosis for chronically infected HCV patients, a high level of HCV-induced disease burden remains due to low rate of diagnosis (% here) and the high cost of therapy. Without treatment, 80% of chronically infected hepatitis C individuals exhibit hepatic intracellular lipid accumulation, termed steatosis. This liver damage can eventually contribute to cirrhosis (20% of individuals) and hepatocellular carcinoma (5% of individuals), necessitating a liver transplant for patient survival. Hepatitis C virus (HCV) infection perturbs host lipid metabolism through both cellular and viral-induced mechanisms, with the viral core protein playing an important role in steatosis development. In the first three years of my Ph.D., I sought to identify mechanisms contributing to HCV-induced steatosis. This work was published and Spotlighted in Journal of Virology. In this study, we identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as a HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. We then further investigated the biological significance and molecular mechanism of this downregulation. Importantly, we validated our in vitro finding with an in vivo model system, and saw that HCV infection in a live mouse model downregulated CIDEB in the liver tissue. We also found that CIDEB gene knockout in a hepatoma cell line resulted in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very low density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis was further supported by the requirement of HCV core and its LD localization for CIDEB downregulation, which utilized a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB. In late 2015, the global scientific community became aware of the emergence and threat of another Flaviviridae virus called Zika virus (ZIKV) in Brazil. The previously obscure ZIKV, which had laid relatively dormant for the previous 70 years, began spreading rapidly through the Western hemisphere, thus prompting the World Health Organization (WHO) to declare a public health emergency in February 2016. In response to the global health emergency posed by the ZIKV outbreak and its link to microcephaly and other neurological conditions, we established a collaboration with Dr. Wei Zheng at the National Center for Advancing Translational Sciences (NCATS) at this time, with whom we together performed a drug repurposing screen of ~6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds. We reported these results in Nature Medicine in August 2016. From this initial research, we identified 37 lead compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. We found that emricasan, a pan-caspase inhibitor, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. We then continued our work to identify additional compounds by refining our high-throughput assay. We developed a high-throughput ZIKV-NS1 based FRET detection assay to rescreen all 6,000 compounds, and then validated 256 hits by a semi-automated viral titer assay we developed in our lab in collaboration with NCATS. From this combinatorial approach, we identified an additional 117 compounds for use in further antiviral development. Among these, we found a conserved role of proteasome inhibitors in inhibiting ZIKV infection, and identified additional lead compounds including emetine, an anti-protozoal small molecule compound. In collaboration with NCATS and Dr. Anil Mathew Tharappel who completed mouse studies, we found that emetine is effective in a live animal model at reducing Zika viral load and likely inhibits viral replication via a direct block on the ZIKV NS5 RNA dependent RNA polymerase. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester 2018. / April 16, 2018. / Antiviral, Drug discovery, Flavivirus, Hepatitis C Virus, Steatosis, Zika virus / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Yi Ren, University Representative; Thomas C. Keller, Committee Member; Fanxiu Zhu, Committee Member; David Meckes, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_654735 |
| Contributors | Lee, Emily M. (Emily Michelle) (author), Tang, Hengli (professor directing dissertation), Ren, Yi (university representative), Keller, Thomas C. S. (committee member), Zhu, Fanxiu (committee member), Meckes, David G. (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Biological Science (degree granting departmentdgg) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text, doctoral thesis |
| Format | 1 online resource (172 pages), computer, application/pdf |
Page generated in 0.0028 seconds