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Investigating The Mechanism Of The Coronavirus Endoribonuclease In Antagonizing Innate Immune Signaling, Matthew Hackbart

Dissertations

Coronaviruses (CoVs) are positive-sense RNa viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease (EndoU) that cleaves RNa in biochemical assays, but the target and function of EndoU activity during viral replication was not known. My work focused on characterizing the functions of EndoU during infection. I report that EndoU is an innate immune antagonist. to function as an immune antagonist, EndoU cleaves the 5'-Poly-Uridines from Negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNa synthesis. Using a virus containing an EndoU catalytic-inactive mutation, I detected …

Distinct Roles For Carbohydrate And Protein Receptors In Coronavirus Infection, Enya Qing

Dissertations

Coronaviruses (CoVs) are common human and animal pathogens. in humans, four endemic CoV species together account for one third of mild respiratory infections worldwide. More severe and frequently fatal respiratory pathologies are caused by recent CoV outbreaks that resulted from occasional zoonotic spillover from animal CoV reservoirs, namely, SARS-CoV in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Because CoVs threaten global health, any chance of relieving CoV's threat on human populations would rely heavily on our understanding of the mechanistic requirements for CoV tropism, whose major determinant is at the level of viral entry. CoVs have evolved to use …

Surveying Host Innate Immune Responses To Interferon Antagonist-Deficient Murine Coronaviruses, Aaron Brian Volk

Master's Theses

Two coronaviruses (CoVs)—severe acute respiratory syndrome (SARS) virus and Middle East respiratory syndrome (MERS) virus—have emerged in the 21st century from animal reservoirs into the human population, each causing an epidemic associated with significant disease and mortality. CoV epidemics are currently only controllable by rigorous public health measures; no targeted therapeutics or vaccines exist to treat or prevent any human CoV infection. One method of generating attenuated CoV strains to be studied as vaccine candidates involves specifically disrupting CoV-encoded interferon (IFN) antagonists, thereby rendering the virus vulnerable to host innate antiviral immunity. Deubiquitinating (DUB) activity encoded within CoV nonstructural protein …

Coronavirus Proteases As Therapeutic Targets: Development Of Biosensors To Detect Inhibition Of Protease Activity And Separation Of The Multiple Functions Of Coronavirus Papain-Like Proteases, Andrew Kilianski

Dissertations

Coronaviruses are important human pathogens and have the potential to severely impact public health on an international scale. The emergence of SARS-CoV and MERS-CoV highlight the need for research to identify antivirals and vaccines against coronaviruses. To develop therapeutics against current and potentially emergent coronaviruses, I utilized two approaches targeting the proteases encoded within all coronaviruses. The papain-like protease and 3C-like protease of coronaviruses are responsible for cleaving viral polyproteins early during infection, and this step is required for viral replication. To quantitatively assess the inhibition by small-molecule compounds on MERS-CoV protease activity, I developed a luciferase-based biosensor to monitor …

Multifunctional Coronavirus Papain-Like Proteases As Targets For Antiviral Therapeutics And Vaccines, Anna Maria Mielech

Dissertations

Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle Ease Respiratory Syndrome coronavirus (MERS-CoV) pose a severe threat to humans because of high mortality. Despite the risk of coronavirus (CoV) emerging in the human population there are no antiviral drugs or vaccines to combat coronavirus infection. The focus of my dissertation was to study the multifunctionality of papain-like proteases (PLPs) encoded within coronavirus genomes to facilitate the development of antiviral drugs and vaccines. The viral PLPs are critical for processing the amino-terminal end of the replicase during virus replication and are attractive targets for antiviral therapies.

In my research, I analyzed …

Coronavirus Replicase Proteins: Multifunctional Mediators Of Replication And Innate Immunity Evasion, Mark Anthony Clementz

Dissertations

Coronaviruses are positive-sense, single-stranded RNA viruses. The majority of the RNA encodes non-structural proteins (nsps) that are translated as a large polyprotein, which is cleaved by the papain-like (PLP) and picornavirus 3C-like (3CLpro) proteases. The nsps modify host membranes to produce double membrane vesicles (DMVs) upon which the replicase-transcriptase assembles and synthesizes viral RNA. nsp3, nsp4, and nsp6 are integral membrane proteins believed to be involved in DMV formation. Work presented here demonstrates that nsp4 is subjected to N-linked glycosylation and mutation of N258 to threonine in nsp4 confers a temperature sensitive phenotype to MHV-A59 infectious clone virus. This virus …