Browsing by Author "Geiss, Brian J., advisor"

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Open Access
Dodging wrenches in the time of COVID: exploring flavivirus replication mechanisms and SARS-CoV-2 antibody development
(Colorado State University. Libraries, 2024) Terry, James Steven, author; Geiss, Brian J., advisor; Wilusz, Jeffrey, committee member; Ebel, Gregory, committee member; Snow, Christopher, committee member
Flaviviruses 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.
Open Access
Molecular diagnostic platforms for point-of-need pathogen detection
(Colorado State University. Libraries, 2021) Jain, Sidhartha, author; Henry, Charles S., advisor; Geiss, Brian J., advisor; Dandy, David S., committee member; Magzamen, Sheryl L., committee member
Rapid, accurate, reliable nucleic acid testing (NAT) platforms are essential in the diagnosis and management of diseases. The inherent complexity associated with NAT requires that such testing be performed in centralized laboratories by highly trained personnel. Modified molecular technologies that can be used at the point-of-care (POC) are needed to improve the turnaround times of results and lower the global burden of infectious diseases. To help address this urgent need, we have developed a nucleic acid sensor platform utilizing nuclease protection and lateral flow detection for rapid, point-of-need nucleic acid analysis. We have also improved the analytical performance of the assay by pairing it with isothermal padlock rolling circle amplification (RCA). RCA is one of the simplest and most versatile isothermal amplification techniques as it only requires one primer and a strand-displacing polymerase. Utilizing our rolling circle amplification lateral flow platform, we have developed assays for beta-lactamase resistance genes for antimicrobial resistance monitoring and severe acute respiratory virus coronavirus 2 (SARS-CoV-2). We have also explored the use of exponential isothermal amplification to further improve the assay limit of detection. We also propose a microfluidic device to rapidly detect the RCA amplicons. The device allows programmable sequential delivery of reagents to a detection region, reducing the number of user steps. With further development, such microfluidic devices can be used to develop fully integrated sample-to-result molecular diagnostic platforms that integrate sample pretreatment, amplification, and detection in an easy-to-use, point-of-need nucleic acid sensor platform. Chapter 1 presents a brief review of the nucleic acid testing landscape, the challenges associated with the development of point-of-need nucleic acid sensors and recent successes utilizing paper-based devices for fully integrated sample-to-result sensors. Chapters 2 and 3 discuss the development of the nuclease protection lateral flow assay and padlock probe-based rolling circle amplification lateral flow assay. Chapter 4 describes our work on the use of exponential RCA to improve the limit of detection of the SARS-CoV-2 assay. In Chapter 5, we present our work on a paper-plastic microfluidic device for the rapid detection of the RCA amplicon. We believe that such devices can be used for the development of integrated molecular diagnostic sensor platforms that can be used at the point-of-need in resource-limited settings.
Open Access
The flavivirus NS3 helicase Motif V controls unwinding function and alters viral pathogenesis in mosquitoes
(Colorado State University. Libraries, 2020) Du Pont, Kelly Elizabeth, author; McCullagh, Martin, advisor; Geiss, Brian J., advisor; Szamel, Grzegorz, committee member; Snow, Christopher, committee member; Krummel, Amber, committee member; Ho, Shing, committee member
Over half of the world's population is at risk of flavivirus (e.g. dengue virus, West Nile virus, Japanese Encephalitis virus, and Zika virus) infection making it a global health concern. These specific mosquito-borne flaviviruses are responsible for causing a variety of symptoms and outcomes including flu-like fevers, encephalitis, hemorrhagic fevers, microcephaly, Guillain-Barré syndrome, and death. Unfortunately, vaccines and anti-viral therapeutics are not always effective in protecting against and treating viral infections. Sometimes these therapies cause more severe symptoms through an antibody dependent enhancement. Therefore, there is a pressing need for the development of effective anti-viral therapies against each flavivirus. For the advancement of these interventional strategies, a fundamental understanding of how flaviviruses replicate within hosts, including the mosquito vector, is required. This dissertation investigates how flaviviruses regulate viral replication, pathogenesis and mosquito transmission through the nonstructural protein 3 (NS3) helicase structure and function. A combination of virology, biochemistry, and computational simulations will be utilized to address how NS3 plays a role in viral infection, viral replication, and viral protein structure. An essential aspect of flaviviral genome replication is the unwinding of the double-stranded RNA intermediate via the C-terminal helicase domain of NS3. NS3 helicase translocates along and unwinds the double-stranded nucleic acids in an ATP-dependent manner. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well understood. Previous simulations in the group led us to hypothesize that Motif V is a critical component of the transduction mechanism. Here, we tested Motif V mutations in both sub-genomic replicon and recombinant protein systems to examine viral genome replication, helicase unwinding activity, ATP hydrolysis activity, and RNA binding affinity activity. NS3 helicase mutants, T407A and S411A, indicated reduced viral genome replication and increased turnover rates of helicase unwinding activity by a factor of 1.7 and 3.5 respectively. Additionally, we simulated Motif V mutants to probe the structural changes within NS3 helicase caused by the mutations. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. Motif V mutations T407A and S411A exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during viral genome replication. Next, we utilized T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo to probe the how these mutations play a role in pathogenesis and transmission of flaviviruses. Of the two Kunjin virus mutants, only S411A Kunjin virus was recovered. In cell culture, S411A Kunjin decreased viral infection and increased cytopathogenicity as compared to WT Kunjin. Similarly, in surviving Culex quinquefasciatus mosquitoes, S411A Kunjin decreased infection rates as compared to WT Kunjin, but S411A Kunjin infection increased mortality compared with that of WT Kunjin infection. Additionally, S411A Kunjin increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin. These data suggest that S411A Kunjin increases pathogenesis in mosquitoes. Overall, these computational simulation, biochemical assay, and virology data indicate that flavivirus NS3 helicase Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus, not just regulation of translocation and unwinding during viral genome replication. The molecular level insights presented in this dissertation provide the fundamental research for understanding how to target specific regions of NS3 helicase for the advancement of anti-viral therapeutics.