Virology Commons^™

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 …

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 …

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 …

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 …

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). …

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 …

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 …

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 …

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 …

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 …

Phenotyping Temperature-Sensitive Coronaviruses, Amani Eddins

Master's Theses

Coronaviruses (CoVs) can cause a range of symptoms; from a mild common cold to life threatening acute respiratory distress syndrome (ARDS) upon infection. These emerging viruses have the ability to be highly pathogenic and detrimental to the human population. Two prime examples of CoV emergence, SARS-CoV (Severe Acute Respiratory Syndrome) and MERS-CoV, (Middle Eastern Respiratory Syndrome), which exhibits the pandemic potential of emerging CoVs that gain human tropism. Given the increasing potential of an emerging CoV outbreak there is a state of urgency to develop vaccines that will help protect the human population against current and future circulating strains. The …

Cellular Determinants Of Coronavirus Entry Routes, James Thomas Earnest

Dissertations

Coronaviruses (CoVs) represent some of the greatest modern threats to global health. CoVs are zoonotic viruses that generally cause respiratory or enteric infections. The ability of CoVs to move between species and into human populations ensures that CoVs will remain important and dangerous pathogens. Therefore, understanding how CoVs infect different hosts is vital to human health.

CoVs are enveloped and must undergo fusion of viral and host membranes to initiate infection. Viral glycoproteins, called Spike (S) proteins, are responsible for host cell binding and carrying out the membrane fusion reaction. S proteins store energy in their folded, pre-fusion conformation that …

The Use Of Informativity In The Development Of Robust Viromics-Based Examinations, Siobhan C. Watkins, Catherine Putonti

Bioinformatics Faculty Publications

Metagenomics-based studies have provided insight into many of the complex microbial communities responsible for maintaining life on this planet. Sequencing efforts often uncover novel genetic content; this is most evident for phage communities, in which upwards of 90% of all sequences exhibit no similarity to any sequence in current data repositories. For the small fraction that can be identified, the top BLAST hit is generally posited as being representative of a viral taxon present in the sample of origin. Homology-based classification, however, can be misleading as sequence repositories capture but a small fraction of phage diversity. Furthermore, lateral gene transfer …

Genomes Of Gardnerella Strains Reveal An Abundance Of Prophages Within The Bladder Microbiome, Kema Malki, Jason W. Shapiro, Travis Kyle Price, Evann Elizabeth Hilt, Krystal Thomas-White, Trina Sircar, Amy B. Rosenfeld, Michael J. Zilliox, Alan J. Wolfe, Catherine Putonti

Bioinformatics Faculty Publications

Bacterial surveys of the vaginal and bladder human microbiota have revealed an abundance of many similar bacterial taxa. As the bladder was once thought to be sterile, the complex interactions between microbes within the bladder have yet to be characterized. To initiate this process, we have begun sequencing isolates, including the clinically relevant genus Gardnerella. Herein, we present the genomic sequences of four Gardnerella strains isolated from the bladders of women with symptoms of urgency urinary incontinence; these are the first Gardnerella genomes produced from this niche. Congruent to genomic characterization of Gardnerella isolates from the reproductive tract, isolates …

Freshwater Metaviromics And Bacteriophages: A Current Assessment Of The State Of The Art In Relation To Bioinformatic Challenges, Katherine Bruder, Kema Malki, Alexandria Cooper, Emily Sible, Jason W. Shapiro, Siobhan C. Watkins, Catherine Putonti

Bioinformatics Faculty Publications

Advances in bioinformatics and sequencing technologies have allowed for the analysis of complex microbial communities at an unprecedented rate. While much focus is often placed on the cellular members of these communities, viruses play a pivotal role, particularly bacteria-infecting viruses (bacteriophages); phages mediate global biogeochemical processes and drive microbial evolution through bacterial grazing and horizontal gene transfer. Despite their importance and ubiquity in nature, very little is known about the diversity and structure of viral communities. Though the need for culture-based methods for viral identification has been somewhat circumvented through metagenomic techniques, the analysis of metaviromic data is marred with …

Phagephisher: A Pipeline For The Discovery Of Covert Viral Sequences In Complex Genomic Datasets, Thomas Hatzopoulos, Siobhan C. Watkins, Catherine Putonti

Bioinformatics Faculty Publications

Obtaining meaningful viral information from large sequencing datasets presents unique challenges distinct from prokaryotic and eukaryotic sequencing efforts. The difficulties surrounding this issue can be ascribed in part to the genomic plasticity of viruses themselves as well as the scarcity of existing information in genomic databases. The open-source software PhagePhisher (http://www.putonti-lab.com/phagephisher) has been designed as a simple pipeline to extract relevant information from complex and mixed datasets, and will improve the examination of bacteriophages, viruses, and virally related sequences, in a range of environments. Key aspects of the software include speed and ease of use; PhagePhisher can be used with …

Adenovirus Evasion Of Cell-Intrinsic Immunity, Andrew Michael Burrage

Dissertations

Virus cell entry represents one of the earliest opportunities for a host to respond to infection. Understanding the processes of pathogen detection and restriction employed by the host, as well as strategies utilized by the virus itself to evade such processes, is critical in developing therapeutics to counter pathogenesis. Adenovirus (Ad) infections are self-limiting in healthy populations, but can be devastating to individuals with compromised immune systems. Currently, no specific antiviral treatments exist to combat Ad infections in susceptible populations. However, because Ad infections are not severe in healthy individuals, employing replication-defective Ads as vaccine vectors is generally regarded as …

Bacteriophages Isolated From Lake Michigan Demonstrate Broad Host-Range Across Several Bacterial Phyla, Kema Malki, Alex Kula, Katherine Bruder, Emily Sible, Thomas Hatzopoulos, Stephanie Steidel, Siobhan C. Watkins, Catherine Putonti

Biology: Faculty Publications and Other Works

BACKGROUND:

The study of bacteriophages continues to generate key information about microbial interactions in the environment. Many phenotypic characteristics of bacteriophages cannot be examined by sequencing alone, further highlighting the necessity for isolation and examination of phages from environmental samples. While much of our current knowledge base has been generated by the study of marine phages, freshwater viruses are understudied in comparison. Our group has previously conducted metagenomics-based studies samples collected from Lake Michigan - the data presented in this study relate to four phages that were extracted from the same samples.

FINDINGS:

Four phages were extracted from Lake Michigan …

The Effects Of Stress And Alcohol On Hiv-1 Latency, Sarah Ilene Talley

Master's Theses

A major barrier to HIV eradication is the persistence of latent viral reservoirs that exist despite antiretroviral therapy. In order to develop effective therapeutics, a comprehensive understanding of latency and factors driving the formation of the latent reservoir is needed. As stress and alcohol are common comorbidities associated with HIV infection, the goal of this research was to determine how stress and alcohol could affect HIV-latency. Specifically, we hypothesized that the ability of latent proviruses to be reactivated by “shock and kill” approaches would be altered by glucocorticoid and ethanol treatments, and prolonged ethanol exposure would affect the size of …

The Role Of Pidd Protein In Adenoviral Induction Of Apoptosis, Iris Teresa Figueroa

Master's Theses

The Adenovirus E1A gene sensitizes cells to genetic insults and apoptosis, most notably in response to cytotoxic factors from innate immune cells. The mechanisms by which E1A sensitizes cells to apoptotic stressors have not been fully elucidated, however E1A actively represses NF-κB anti-apoptotic defenses, thereby sensitizing cells to alternative apoptotic cascades. Recent studies alternatively indicate that E1A also actively induces Caspase-2 activation and mitochondrial injury in the presence of certain cytotoxic injuries.

Caspase-2 is cleaved and activated in a large complex consisting of p53-inducible protein with a death domain (PIDD), an adaptor protein RAIDD, and pro-caspase 2. PIDD auto-proteolyzes into …

Malaria Transmission Blocking Vaccines, Lukasz Jacek Sewera

Master's Theses

Malaria affects 198 million people and kills 584,000 each year, predominantly in Sub-Saharan Africa (WHO). The most severe form of malaria is caused by the protozoan parasite Plasmodium falciparum. Development of a vaccine against P. falciparum has been hindered by its complex life cycle with multiple antigenically distinct human and mosquito stages. To effectively prevent disease and reduce the parasite burden in populations, a vaccine will need to target multiple stages, including blocking transmission at the mosquito stage.

Antibodies generated against P. falciparum mosquito stage antigen Pfs25 can prevent parasite transmission from humans to mosquitoes. However, Pfs25 is poorly immunogenic …