Virology Commons^™

Investigating Host Factors In Coronavirus Assembly And Egress, Surovi Binte Sharmin Mohona

Dissertations

Coronavirus (CoV) structural proteins congregate at the ER-Golgi intermediate compartments (ERGIC) prior to virus budding into the organelle lumen. Subsequent trafficking and fusion of virus-containing, ERGIC-derived vesicles then release nascent viruses from infected cells. Our findings in this dissertation illuminate mechanisms by which CoV structural proteins coalesce at the virus assembly and budding sites. The cytoplasmic tails of all CoV spike (S) glycoproteins contain variants of a consensus KxKxx coatomer-binding motif. Host coatomer complexes facilitate retrograde trafficking of client cargo proteins via recognition of such motifs. Utilizing reverse genetics methodologies, we introduced variations in the S coatomer motif in a …

Investigating The Consequences Of Conjugated And Secreted Isg15 On Coronavirus Pathogenesis, Mudassir Syed Ali

Dissertations

Coronaviruses have evolved a breadth of mechanisms to antagonize host innate immune responses. One widely studied mechanism is the ability to deconjugate ISG15 from target proteins in a process called deISGylation, which is catalyzed by the papain-like protease (PLP) domain of viral non-structural protein (nsp) 3. Since PLP activity is required for viral replication, it has been challenging to generate an infectious virus with a PLP that maintains protease activity but has lost deISGylase activity. In this study, we generated a chimeric MHV-A59 with its PLP2 domain replaced with that of the human coronavirus HKU1, which has been documented to …

Investigating The Proviral And Immunogenic Effects Of Sars-Cov-2 Accessory Protein 7a, Grant Michael Hawkins

Dissertations

SARS-CoV-2 and related sarbecoviruses encode a set of accessory proteins (3a, 3b, 6, 7a, 7b, 8, 9b, and 10) that control host responses to infection and promote virus growth. Of these accessory proteins, 7a is set apart by its intracellular localization near coronavirus (CoV) budding sites and its incorporation into secreted virions. Initial studies utilizing a virus-like particle (VLP) system identified significant proviral effects mediated by 7a, but the system lacked the context of an actual CoV infection. To investigate 7a functions during CoV infections, we constructed recombinant Mouse Hepatitis Viruses (rMHV strain A59) that express 7a genes. Comparative infections …

Discerning The Role Of Microtubule Acetylation During Hiv-1 Infection, Drew Michael Lichon

Dissertations

The host-pathogen interactions underlying Human Immunodeficiency Virus type 1 (HIV-1) infection are complex and involve the exploitation of host cellular machinery. This thesis investigates the roles of dynein adaptors, microtubule acetylation, and TRIM69-mediated restriction in HIV-1 infection. Through a series of detailed experiments, we explore how HIV-1 utilizes multiple dynein adaptors, such as BICDR1 and Hook3, for intracellular transport, and reveal that these adaptors are critical for efficient viral trafficking. Conversely, the knockdown of Ninein surprisingly enhances HIV-1 infectivity, suggesting a nuanced regulatory role for this adaptor in viral transport. Further, this thesis challenges the prevailing paradigm that microtubule acetylation …

Investigating The Role Of The Nsp15 Amino-Terminal Domain In Nsp15 Function, Viral Replication, And Pathogenesis, Monika Evdokimova

Dissertations

Identifying conserved mechanisms used by viruses to delay host innate responses can reveal potential targets for antiviral therapeutics. Here, we investigated coronavirus nonstructural protein 15 (nsp15), which encodes a highly conserved endoribonuclease (EndoU). EndoU functions as an immune antagonist by limiting the accumulation of viral replication intermediates that would otherwise be sensed by the host. Despite being a promising antiviral target, it has been difficult to develop small molecule inhibitors that target the EndoU active site. We generated nsp15 mutants of SARS-CoV-2 and mouse coronavirus MHV-A59 and identified conserved residues within the amino-terminal domain that are required for EndoU activity. …

Role Of Coronavirus Envelope Protein On Infectivity, Garrett E. Zaagman

Dissertations

Coronaviruses infect a wide range of hosts and have caused recent human pandemics. While much has been learned, gaps remain in understanding viral assembly and egress, particularly the role of the envelope (E) protein in specific infectivity. To study this, recombinant MHV viruses were created with E gene deletions and HiBiT-tagged spike (S) proteins to monitor virion release. In mouse cells, MHV∆E S-HiBiT secreted less spike and fewer infectious particles, indicating reduced specific infectivity. Comparisons between human HeLa-CEACAM and mouse DBT cells showed species-specific support for virus production. TMED10, a host membrane protein, was tested as a potential E-interacting partner …

Investigating The Roles Of Sirtuins During Cvb3 Infection And Innate Immunity, Bridget M. Hulsebosch

Master's Theses

Coxsackievirus B3 (CVB3) is an enterovirus that chronically infects heart tissue, leading to dilated cardiomyopathy. However, no vaccines or antivirals exist for CVB3 infection or to treat CVB3-induced dilated cardiomyopathy, except for a heart transplant. Sirtuins (SIRTs), class III lysine deacetylases, have diverse and complex roles in cellular functions, contributing to energy production, NF-κB signaling, cell cycle regulation, and inflammation. Previous studies showed that SIRTs are essential for hepatitis B and A viral replication and regulate the IFN response during viral infection. However, the role of SIRT1 and 2 during CVB3 infection is entirely unknown. CVB3-infected SIRT1 and SIRT2 knockout …

Novel Antivirals Unveiled Against Acute And Persistent Coxsackievirus B3 Infection Via Rapid Screening, Maria Del Mar Villanueva Guzman

Master's Theses

Enteroviruses cause 10 to 15 million infections annually in the United States, and Coxsackievirus B3 is one of the most commonly reported. Coxsackieviruses may become persistent, characterized as a viral infection that is not cleared from host cells and that generates a continuous infection. Patients who develop persistent CVB3 infection may not respond to the same antivirals as an acute infection, which may be detrimental. Therefore, there is a need for broad-range antiviral drugs to combat acute and persistent CVB3 infection, as there is no well-accepted treatment available. We developed a model system to study persistent CVB3 using a pancreatic …

Novel Antivirals Unveiled Against Acute And Persistent Coxsackievirus B3 Infection Via Rapid Screening, Maria Del Mar Villanueva Guzman

Master's Theses

Enteroviruses cause 10 to 15 million infections annually in the United States, and Coxsackievirus B3 is one of the most commonly reported. Coxsackieviruses may become persistent, characterized as a viral infection that is not cleared from host cells and that generates a continuous infection. Patients who develop persistent CVB3 infection may not respond to the same antivirals as an acute infection, which may be detrimental. Therefore, there is a need for broad-range antiviral drugs to combat acute and persistent CVB3 infection, as there is no well-accepted treatment available. We developed a model system to study persistent CVB3 using a pancreatic …

Elucidating The Underlying Mechanisms Polyamines Play On Coxsackievirus B3 Infection, Mason Richard Firpo

Dissertations

Polyamines are a class of metabolites consisting of small carbon chains that contain amine groups and are positively charged at cellular pH. They play many important roles within the cell and are necessary for cell growth, membrane function, transcription, and translation. They are also required by a myriad of viruses for a productive infection. This includes the non-polio enterovirus Coxsackievirus B3 (CVB3). CVB3 is a cardiotropic virus that can readily infect the heart and cause irreversible damage which can eventually lead to dilated cardiomyopathy and the need for a heart transplant. Unfortunately, there are no approved antivirals or vaccines that …

Establishing A Novel Lipid-Polyamine Metabolic Connection To Virus Replication, Yazmin Edith Cruz Pulido

Dissertations

Polyamines are small positively charged alkylamines found in all living cells. These ubiquitous molecules have been found to play a variety of roles ranging from stabilizing nucleic acids (DNA and RNA) to cellular proliferation. Studies have also identified roles for polyamines in viral packaging and viral enzyme function. However, the role of polyamines on cellular components and their effect on viral replication is less known. In our efforts to identify the lipid-polyamine metabolic connection to virus replication we utilized a human hepatocyte cell line in which polyamines were depleted via pharmacological or genetic approaches and studied the effects on lipid …

Role Of Tetraspanins In Sars-Cov-2 Fusion And Entry, Marcos Saul Santiago Figueroa

Master's Theses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic the world has been facing in recent years. Even as new countermeasures are developed, there is still much we don’t know in terms of its entry into host cells. Tetraspanins are transmembrane proteins that are near ubiquitous amongst cell types. They fulfill numerous roles, including that of a viral co-receptor. Here, we considered whether tetraspanins, specifically CD9, CD63, and CD81 influence SARS-CoV-2 fusion and entry. Using ACE2-LgBit and tetraspanin overexpressing EVs and HeLa cells, we find that the presence of excess tetraspanins inhibit fusion and entry. However, we …

Bisacodyl Limits Chikungunya Virus In Vitro And Is Broadly Antiviral, Natalie June Lomascolo

Master's Theses

Identifying novel antivirals requires significant time and resource investment, and the continuous threat of viruses to human health necessitates commitment to antiviral identification and development. Developing antivirals requires years of research and validation, and recent outbreaks have highlighted the need for preparedness in counteracting pandemics. One way to facilitate development is to repurpose molecules already used clinically. By screening such compounds, we can accelerate antiviral development. Here, we screened compounds from the National Institutes of Health’s Developmental Therapeutic Program for activity against chikungunya virus, an alphavirus that is responsible for a significant outbreak in the Americas in 2013. Using this …

Characterizing A Novel Cocksackievirus B3 Protease Mutant And Its Response To Polyamine Depletion, Bridget Hulsebosch

Master's Theses

Enteroviruses, including Coxsackievirus B3 (CVB3), are pervasive pathogens that cause significant disease, including cardiomyopathies. Unfortunately, no treatments or vaccines are available for infected individuals. We identified the host polyamine pathway as a potential drug target, as inhibiting polyamine biosynthesis significantly reduces enterovirus replication in vitro and in vivo. Here, we show that CVB3 is sensitive to polyamine depletion through the polyamine analog diethylnorspermidine (DENSpm) which enhances polyamine catabolism through induction of polyamine acetylation. We demonstrate that CVB3 acquires resistance to DENSpm via mutation of the 2A protease, which enhances proteolytic activity in the presence of DENSpm. Resistance to DENSpm occurred …

Assessing The Role Of The Interaction Between Polyamines And Heparan Sulfate In Coxsackievirus B3 Viral Infection, Oreoluwa Omoba

Master's Theses

Non-polioenteroviruses are highly infectious viruses that typically cause mild asymptomatic cases but can cause severe disease such as aseptic meningitis and dilated cardiomyopathy. Coxsackievirus B3 (CVB3) is a member of this group and is prevalent in the United States and Eastern Asia. Viruses like CVB3 rely on the host cell for many molecules in order to replicate. One such set of molecules is the polyamines. Polyamines are small, positively charged molecules that play a role in a multitude of cellular processes including RNA/DNA stabilization, gene expression, translation, and regulating membrane fluidity. Previous studies have shown CVB3 relies on polyamines for …

Zika Virus Inactivates Polyamine Catabolism Via Alternative Splicing To Enhance Infection, Marion Lea Graham

Master's Theses

During viral infection, the virus and host must compete for resources inside the cell. One of these resources is polyamines. Polyamines are small, positively charged molecules that are found in all eukaryotic cells. They play a key role in several cellular functions including growth and proliferation, transcription and translation, and membrane stability. Viruses also rely polyamines for productive replication, utilizing them during DNA/RNa polymerization, nucleic acid packaging, and protein synthesis. in response to a virus infecting a host cell, the host cell will begin to regulate polyamine levels as a way to combat the infection. Polyamine levels are regulated by …

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 …

Elucidating The Impact And Mechanism Of Hepatitis C Virus Cell-To-Cell Spread, Karina Durso-Cain

Dissertations

Virus cell-to-cell spread has been implicated in the establishment of persistent infection and has been shown to be involved in the transmission of antiviral resistant mutants. However, relatively little is known about how virus cell-to-cell spread impacts infection or the specific mechanisms by which cell-to-cell spread occurs. as such, this dissertation focused on investigating HCV cell-to-cell spread not only to learn more about this medically important virus, but also to determine the broader impact cell-to-cell spread has on viral infection dynamics, identify the cellular factors involved, and perhaps ultimately provide insight into antiviral strategies that might enhance the barrier to …

Dissecting A Role For Polyamines In Rift Valley Fever Virus Infection, Vincent Mastrodomenico

Master's Theses

Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. Rift Valley fever virus is a particularly devastating bunyavirus, infecting both humans and livestock with significant morbidity and mortality. By coordinating several host and viral processes Rift Valley fever virus is able to produce infectious virions. Polyamines are small, positively-charged host-derived molecules that play diverse roles in human cells and in infection. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated polyamines' role in the replication of the Rift Valley fever virus (vaccine strain MP-12). …

The Role Of Polyamines In Enteroviral Attachment, Thomas Mckenzie Kicmal

Master's Theses

Polyamines are small polycationic molecules with flexible carbon chains that are found in all eukaryotic cells. Polyamines are involved in the regulation of many host processes and have been shown to be implicated in viral replication. Depletion of polyamine pools in cells with FDA approved drugs restricts replication of diverse RNA viruses. Viruses can exploit host polyamines to facilitate packaging, transcription, translation, and protease activity but other mechanisms remain largely unknown. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to depletion of polyamines and remain a significant public health threat. We employed CVB3 as a model system to investigate a potential …

Membrane Microdomains As Platforms For Extra-Cellular Fusions, Michael Hantak

Dissertations

Life requires biological membranes. Membrane-enclosed compartments separate and unite through dynamic fission and fusion reactions. These are catalyzed processes that are central in organismal biogenesis. This dissertation focuses on extracellular membrane fusions, which are central to several processes. (1) Enveloped viruses enter cells through membrane fusions. (2) Extracellular vesicles (EVs) also deliver molecules into cells through membrane fusions. (3) Entire cells also fuse together, generating fertilized zygotes, skeletal muscles, and giant cell macrophages.Mechanisms of extracellular membrane fusion are poorly understood. This dissertation aimed to further define these mechanisms. We focused on regulatory cofactors, including tetraspanins, transmembrane proteins that cluster into …

Generating Biologic And Genetic Research Tools To Investigate Serotype I Feline Coronaviruses, Robert Christian Mettelman

Dissertations

Serotype I feline infectious peritonitis virus (FIPV) is an alphacoronavirus of high veterinary importance due to the 99% mortality rate observed in cats. Since the 1980s, robust experimentation on these viruses has been limited by availability of culturable pathogenic strains, few permissive cell lines, and a lack of standardized methods to study their basic virology. These complications have resulted in variable and conflicting literature reports, have slowed study of clinical strains and hindered effective vaccine design. The goal of this dissertation was to develop a laboratory "toolbox" containing standardized methods, permissive cell lines and genetic techniques to alleviate some of …

The Evolution Of Molecular Compatibility Between Bacteriophage Φx174 And Its Host, Alexander Kula, Joseph Saelens, Alyxandria M. Schubert, Michael Travisano, Catherine Putonti

Bioinformatics Faculty Publications

Viruses rely upon their hosts for biosynthesis of viral RNA, DNA and protein. This dependency frequently engenders strong selection for virus genome compatibility with potential hosts, appropriate gene regulation and expression necessary for a successful infection. While bioinformatic studies have shown strong correlations between codon usage in viral and host genomes, the selective factors by which this compatibility evolves remain a matter of conjecture. Engineered to include codons with a lesser usage and/or tRNA abundance within the host, three different attenuated strains of the bacterial virus ФX174 were created and propagated via serial transfers. Molecular sequence data indicate that biosynthetic …

Gene Co-Occurrence Networks Reflect Bacteriophage Ecology And Evolution, Jason W. Shapiro, Catherine Putonti

Biology: Faculty Publications and Other Works

Bacteriophages are the most abundant and diverse biological entities on the planet, and new phage genomes are being discovered at a rapid pace. As more phage genomes are published, new methods are needed for placing these genomes in an ecological and evolutionary context. Phages are difficult to study by phylogenetic methods, because they exchange genes regularly, and no single gene is conserved across all phages. Here, we demonstrate how gene-level networks can provide a high-resolution view of phage genetic diversity and offer a novel perspective on virus ecology. We focus our analyses on virus host range and show how network …

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 …

Immunobiology Of Adenovirus-Vector Vaccines For Mrsa, Emily Orvis

Master's Theses

Staphylococcus aureus is a gram-positive, extracellular bacterium that has emerged as an

important human pathogen. This bacterium is a leading cause of skin and soft tissue infections

(SSTIs) in humans, often leading to invasive and life-threatening infections. Treatment of S.

aureus infections is becoming more complicated due to the rise of methicillin-resistant S. aureus

(MRSA) strains, which are becoming increasingly resistant to a number of antibiotics. In the

United States, invasive MRSA infections result in more deaths annually than any other infectious

agent.

Despite a dire need, there is currently no vaccine against S. aureus infections. The failure

of past …

Characterizing Immune Response To Hiv-1 Infection In Bicd2-Knockout Cells, Omar Abdel-Rahim

Master's Theses

An important part of the HIV-1 infection cycle is the attachment of the intracellular viral core to the host microtubule network, facilitated by attachment of the viral capsid to cargo adaptor proteins. One such cargo adaptor is Bicaudal D Homolog Protein 2 (BICD2). BICD2 can attach to both the HIV-1 capsid and the dynein/dynactin complex and facilitate the trafficking of the viral core towards the host nucleus. Removal of BICD2 can disrupt this viral translocation, resulting in an elevated immune response that impairs productive HIV-1 infection. In my research, we investigated what viral particles are detected in the absence of …

Molecular Determinants Of Trim5Α Restriction And Recruitment Of Autophagic Effectors, Sabrina Imam

Dissertations

TRIM5α is an anti-viral restriction factor that inhibits the lifecycle of retroviruses. TRIM5α binds to and forms a hexameric lattice around the retroviral capsid, thereby initiating its antiviral activities, which include: (1) inhibition of viral infection; (2) inhibition of viral reverse transcription; (3) disassembly of the capsid; and (4), activation of innate signaling pathways. The formation of this assembly also activates the E3 ubiquitin ligase function of TRIM5α. Ubiquitin modification is associated with directing substrates to particular cellular pathways. We and others have shown that TRIM5α cytoplasmic bodies colocalize with proteins involved in the autophagy pathway, and we hypothesized that …

Uncovering The Mechanisms Underlying The Immunogenicity Of Adenovirus Vaccine Vectors, Natalie Nidetz

Dissertations

Vaccination is historically the most effective tool for preventing infectious disease but current vaccine strategies fail to generate robust immunity to major infectious diseases such as HIV and malaria. Therefore, newer vaccine approaches are needed. Vaccines generated from viral, adenovirus based, vectors (AdVs) have proven highly immunogenic in multiple disease models. However, the clinical use of many AdVs is limited by the presence of pre-existing antibodies in human populations, which prevent expression of antigenic genes during immunization with AdVs based on common adenovirus (Ad) serotypes, such as HAdV-5C. Immunization with rare serotype based AdVs, such as HAdV-28D, are not affected …