Terry, James Steven, authorGeiss, Brian J., advisorWilusz, Jeffrey, committee memberEbel, Gregory, committee memberSnow, Christopher, committee member2024-12-232024-12-232024https://hdl.handle.net/10217/239838Flaviviruses pose a significant threat to global health, threatening hundreds of millions of people who live in endemic areas. Infection with flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV) can trigger symptoms ranging from a mild cold-like illness to microcephaly, encephalitis, hemorrhagic fever, and death. As climate change alters global temperature ranges, habitable environments for the flavivirus arthropod vectors are expanding into previously unexposed regions. Due to a lack of flavivirus vaccines and antivirals, most efforts to combat infection fall within vector population control and palliative care for infected individuals. To develop antivirals and vaccines against flaviviruses, we need to better understand the fundamental mechanisms through with the viruses replicate. By investigating incompletely understood processes in the replication cycle, new antiviral targets can be identified and pursued. This dissertation investigates two components of the flavivirus replication cycle to better understand the key processes necessary for successful flavivirus infection. Additionally, this dissertation reports on efforts during the SARS-CoV-2 COVID-19 pandemic to develop novel reagents to assist in research and diagnostic development. An important determinant of successful flavivirus infection is the generation of subgenomic flavivirus RNA (sfRNA). This RNA is composed of exoribonuclease resistant RNA (xrRNA) structures in the flavivirus 3' untranslated region (3'UTR). These structures allow the flavivirus 3'UTR to withstand degradation by stalling the host Xrn1 exoribonuclease, halting viral RNA degradation and creating sfRNA. The production of sfRNA is critical for flavivirus replication success as the new RNA entity actively suppresses the host cell immune response to viral infection. There are blind spots in our understanding of the key stages of sfRNA generation, namely how the Xrn1 substrate is produced from the flavivirus genome. It has previously been postulated that host decapping enzymes remove the flavivirus Type 1 cap structure, allowing Xrn1 to bind to the 5' monophosphate and degrade the viral RNA. The enzyme responsible for decapping has not yet been identified. Following preliminary evidence from the Geiss Lab, we investigated the host decapping enzyme Dcp2 as the protein responsible for priming flavivirus RNA for Xrn1 degradation and sfRNA production. We developed a pipeline using splint-ligation to specifically label monophosphorylated WNV RNA with an RNA adapter at the 5' end. Following this the ratio proportion of viral RNA that is monophosphorylated is revealed using a qRT-PCR reporting system. With this pipeline, it was determined that suppressing Dcp2 expression increased the proportion of monophosphorylated WNV RNA in infected cells while having no significant effect on monophosphorylated RNA in newly produced virions. Additionally, northern blot analysis revealed that sfRNA generation was not reduced by Dcp2 knockdown. From this study we determined that Dcp2 is not necessary for sfRNA generation, and thus other processes are responsible for the generation of monophosphorylated viral genomes for Xrn1 degradation. One hole in our understanding of flavivirus replication concerns the viral replication compartment. The compartment is an invagination in the endoplasmic reticulum membrane that is formed through viral protein manipulation. This environment then hosts the viral replication machinery, protecting the vulnerable viral RNA from host cell immune detection as new viral genomes are produced. Proper viral protein-protein interactions are critical for the successful formation of this viral RNA factory. While studies have been conducted to determine the replication compartment location and some interactions between nonstructural proteins, our understanding of how these proteins interact with each other in situ is limited. To address this, we employed crosslinking mass spectrometry. First, a flavivirus replication compartment purification and crosslinking pipeline underwent a series of evolutions and significant optimizations followed mass spectrometry data acquisition. Then, a crosslinked protein analysis pipeline using the Bonvin Lab programs DisVis/HADDOCK was validated with crosslinked bovine serum to ensure its utility with crosslinked viral compartment samples. MaxQuant analysis revealed some viral protein crosslinks while highlighting areas for improvement in our methodology. Nevertheless, the identified intramolecular crosslinks within NS1, NS3, and NS5 hint at potential dimer interfaces. An intermolecular crosslink between NS3 and NS4b was identified that supports the observations of previous studies while establishing in situ evidence for interactions along an NS3 N-terminus and NS4b residue K172 interface. The results provided intriguing preliminary evidence for future investigations into the replication compartment protein-protein interactions and established a protocol for analyzing viral proteins with crosslinking mass spectrometry. In addition to chronicling on flavivirus replication cycle studies this dissertation includes a chapter chronicling work during the COVID-19 pandemic on SARS-CoV-2. Monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid protein were generated, characterized, and sequenced during the height of the pandemic. These antibodies were the first of their kind to be published and were made available for use during the global SARS-CoV-2 research effort. This chapter also reports on collaborative efforts surrounding the use of antibodies for diagnostics and predictive computational pipelines. Work was done to assist the Henry Lab in developing inexpensive electrochemical and colorimetric ELISA devices targeting SARS-CoV-2 NP for bedside diagnostic use. Lastly, wet lab verification was performed to validate Jacob Deroo's epitope-predicting PAbFold AlphaFold2 pipeline. The work covered in this dissertation spans five years, two viruses, and three separate target areas. These projects, while varied, are all bound together by the common goal of contributing to the advancement of knowledge and techniques for stopping viral threats to global health. Knowing how a virus creates a safe environment for genome replication or identifying which host proteins help create an immune-modulating viral RNA molecule is important for identifying new paths towards intelligently designed antivirals. Similarly, developing and characterizing antibodies to supply a global research effort and validate cutting-edge computational tools is necessary for actively combatting a global pandemic and preparing for the next one. With this work, scientific inquiry ranging from foundational knowledge to translational science is explored.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. 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