Virology Commons^™

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 …

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 …

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 …

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 …

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 …

Reconsidering The Model Of Trim5Α Assembly: The Role Of The Linker2 (L2) Region In Trim5Α Assembly, Laura Johnsen

Master's Theses

The TRIM5α protein from rhesus macaques (TRIM5αrh) exhibits a remarkable ability to potently inhibit infection by Human Immunodeficiency Virus Type-1 (HIV-1). Extensive studies have shown that TRIM5α is capable of self-associating at many levels, eventually leading to the formation of a hexameric assembly that can superimpose on the hexameric lattice of the HIV-1 capsid. The mechanism underlying the self-association of TRIM5α and the molecular determinants of self-association remain to be completely understood. In this study, we show that the Linker 2 (L2) region of TRIM5rh is important for dimerization and higher order self-association, both of which are independent processes. Additionally, …

Host Entry Factors For Human Coronaviruses, Taylor Heald-Sargent

Dissertations

Coronaviruses infect a diverse range of animals from birds to pigs and cats to humans. Coronaviruses employ RNA-based replicative processes and as such are genetically adaptable to acquire novel host ranges. A coronavirus from one species can jump to another by shifting its entry requirements. As we learned from the Severe Acute Respiratory Syndrome (SARS) pandemic, this shift in species can have detrimental consequences. Thus, it is imperative that we understand the virus-host interaction during the entry process.

This dissertation focuses on host entry factors that influence human coronavirus entry. Recently, a new class of proteases, the type II transmembrane …

Adenovirus Vectors As Potent Adjuvants In Vaccine Development, Kathleen Ann Mcguire

Dissertations

Due to their ability to activate the immune system, replication-defective Adenoviruses (Ad) are potential vaccine vectors for several pathogens. The proinflammatory response to Ad contributes to the response to vaccine antigens. We found that reactive oxygen species (ROS) are an important signal in the proinflammatory response to Ad. We identified that serotype 5 adenovirus (Ad5) elicits ROS by inducing mitochondrial membrane damage, a process that is dependent on endosomal membrane rupture and Cathepsin release. This mitochondrial dysfunction contributes to NLRP3 inflammasome- and NFκB-dependent innate immune activation. The ROS-dependent inflammatory response likely contributes to the adaptive immune response by supporting DC …

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 …

Host Proteins Interact With The Hiv-1 Core To Facilitate And Restrict, Zana Lukic

Dissertations

Host cell proteins, termed restriction factors, which inhibit viral replication at various stages of the viral life cycle, determine the species-specific tropism of numerous retroviruses. Many members of the TRIM family of proteins act as viral restriction factors. One well-characterized example is the ability of TRIM5á from rhesus macaques (rhTRIM5á) to inhibit human immunodeficiency virus type-1 (HIV-1) soon after viral entry but prior to reverse transcription (RT). It is well established that the restriction requires an interaction between the viral capsid lattice and the B30.2/SPRY domain of TRIM5á. Following the binding of the viral core, TRIM5á mediates an event or …

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 …

Identifying And Characterizing The Degradative Pathway Of The Retroviral Restriction Factor Rhtrim5Α, Rachel Nelson

Master's Theses

Human immunodeficiency virus 1 (HIV-1) is a lentivirus that progresses to acquired immunodeficiency syndrome (AIDS). The protein TRIM5alpha from rhesus macaques (rhTRIM5alpha) restricts HIV-1 by blocking infection after entry of the virion into cells. Treatment of rhTRIM5alpha expressing cells with inhibitors to a cellular degradation pathway, the proteasome, partially relieves restriction but does not inhibit rhTRIM5alpha protein turnover. The role of a second degradation pathway, the autophagy-lysosomal pathway, in TRIM5alpha mediated restriction has not been explored.

In the present study, we demonstrate that rhTRIM5alpha is degraded by chaperone mediated autophagy (CMA). Inhibition of CMA alters rhTRIM5alpha localization and turnover, while …

Exosomes: Antiviral Agents In The Human Lung, Jennifer Alexandra Novak

Master's Theses

Hundreds of interferon effectors comprise the immediate host response to virus infection. One family of interferon stimulated genes is the IFITM (Interferon-Induced Transmembrane) proteins. IFITM3, in particular, has been shown to block virus cell entry of Influenza A Virus, SARS Coronavirus, and West Nile Virus, among others. IFITM3 has also been identified as an essential factor for host protection against Influenza A Virus in mice and humans. However, the mechanism by which IFITM3 functions remains unclear.

IFITM3 may function both intracellularly and extracellularly to block virus infection in the human lung. The extracellular milieu contains microscopic vesicles termed exosomes which …

Evading Innate And Adaptive Immunity During Adenovirus Cell Entry, Shauna Marvin

Dissertations

Adenovirus (Ad), a non-enveloped, dsDNA virus, enters cells via clathrin-mediated endocytosis. For viral genome delivery to the nucleus, Ad must penetrate endosomal membranes to create defects sufficient for the passage of the 90 nm diameter capsid across cell membranes. Recent observations suggest that adenovirus type 5 (Ad5) capsid uncoating occurs at the cell surface upon binding to both the coxsackievirus and adenovirus receptor and α_v integrins. This uncoating event leads to the exposure of the capsid membrane lytic protein VI. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture, we demonstrate that Ad5 membrane rupture occurs …

The Role Of Linker 2 (L2) Region In Rhtrim5Α Assembly And Hiv-1 Restriction, Jayalaxmi Sastri

Dissertations

The cellular restriction factor TRIM5alpha inhibits infection by numerous retroviruses in a species specific manner. TRIM5alpha protein from rhesus macaques (rhTRIM5alpha) and a related protein TRIM-Cyp from Owl monkeys restrict infection by HIV-1 while human TRIM5α (huTRIM5alpha) restricts infection by N-tropic murine leukemia virus (N-MLV) but not HIV-1. Several models have been proposed for retroviral restriction by TRIM5 proteins (TRIM5alpha and TRIMCyp). These models collectively suggest that TRIM5 proteins mediate restriction by recognizing specific determinants in the viral capsid and directly binding the capsid. Following binding, the TRIM5 proteins self-associate into large assemblies around the viral capsid, which leads to …

Role Of Type Ii Transmembrane Serine Proteases In Coronavirus Production, Gitanjali Cuckemane Subramanya

Master's Theses

Proteolytic cleavage of Coronavirus spike proteins at the appropriate time and location results in efficient virus entry activation. Type II transmembrane serine proteases (TTSPs), specifically, transmembrane protease serine 2 (TMPRSS2) when expressed on target cells, enhance SARS coronavirus entry by activating spike cleavage. This study investigated the effect of TTSPs expressed in Coronavirus producer cells. Murine Hepatitis Virus strain A59 (MHV A59) viruses produced in the absence of TMPRSS2 required target cell protease activity - either cell surface serine proteases or endosomal acidophilic proteases - for entry. MHV A59 viruses produced in the presence of TMPRSS2 were less infectious overall, …

Mechanisms Of Adenovirus Membrane Permeabilization, Oana Maier

Dissertations

For a successful infection to occur, a virus must first penetrate host cell membranes to access intracellular sites of viral replication. Currently the mechanism through which adenovirus, a non-enveloped, dsDNA virus, disrupts the endosomal membrane during cell entry is not well characterized.

Recent studies suggest that adenovirus protein VI, which is released from the interior of the capsid during cell entry, has all of the in vitro membrane lytic activity of the virion. We found that protein VI binds membranes via an amino-terminal 80 residue α-helical domain. Critical to this interaction are conserved hydrophobic and basic lysine residues within this …

Cofactors In Coronavirus Entry, Ana Shulla

Dissertations

Viruses have evolved complex ways to penetrate host barriers and cause disease. One of the most important barriers the virus has to cross is the cellular membrane. Enveloped viruses accomplish this task by viral glycoprotein-mediated binding to host cells and fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. In my dissertation research I focused on understanding the coronavirus spike proteins as well as other cofactors required for S-mediated entry into cells.

The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated …

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 …

The Inhibitory Effects Of The Extracts Of Zingiber Plants On The Adsorption, Growth, And Replication Of Phage Lpp-1 In Cyanobacterium, Ebby Paul Jido

Master's Theses

No abstract provided.