My thesis project explores mechanisms of innate immune evasion by human metapneumovirus (HMPV), as well as the consequences of a lack of type I IFN (IFNAR) signaling on host response and pathogenesis. I show that the expression of the small hydrophobic (SH) of HMPV inhibits type I IFN signaling through targeting signal transducers and activators of transcription 1 (STAT1). Next, I demonstrate that IFNAR signaling is important for the early control of HMPV replication and the development of functional CD8+ T cell (TCD8) response, but it also makes a significant contribution to disease pathogenesis. The effect on a functional T cell response depends on signaling in the bone-marrow compartment, while signaling on the lung epithelium drives pathogenesis. Additionally, the development of decreased functionality in IFNAR-/- mice correlates with expression of the inhibitory T cell receptor T cell immunoglobulin mucin-3 (TIM-3), and a greater ratio of alveolar macrophages to dendritic cells (DCs) early in infection. Taken together, I have revealed mechanisms by which HMPV is capable of evading type I IFN, and I have further elucidated the implications of IFNAR signaling evasion on the TCD8 response.

Chikungunya virus (CHIKV) is a mosquito-borne cause of epidemics of debilitating arthritis worldwide. Currently, there are no licensed vaccines or antiviral therapies available for the prevention or treatment of CHIKV disease. Furthermore, the viral and host determinants required for CHIKV replication and pathogenesis are not understood. Using chimeric viruses generated from virulent and attenuated CHIKV strains, I identified a single residue in the E2 viral attachment protein that serves as a critical determinant of CHIKV replication in cultured cells and pathogenesis in mice. I demonstrate that E2 residue 82 differentially influences infectivity in mammalian and mosquito cells and contributes to interactions with glycosaminoglycans (GAGs). Moreover, mice inoculated with GAG-dependent viruses displayed reduced inflammation in the joint and dissemination to sites of secondary replication. These findings indicate that E2 residue 82 modulates CHIKV dissemination and arthritis and suggest a function for GAG utilization in CHIKV attenuation. Using chemical compound and RNA interference screening approaches, I identified digoxin, an inhibitor of the sodium-potassium ATPase, and PSME2, a regulator of the immunoproteasome, as antagonists of CHIKV infection. Digoxin inhibited CHIKV at both entry and post-entry steps in the replication cycle and was effective against other alphaviruses. Knockdown of PSME2 or blockade of proteasome activity enhanced CHIKV infection. These data suggest roles for the sodium-potassium ATPase and immunoproteasome in CHIKV replication. Collectively, this work reveals mechanisms of CHIKV virulence and provides new targets for the development of CHIKV-specific and broad-spectrum antivirals.

Virus-receptor interactions govern the susceptibility of cells to infection and dictate viral tropism in the host. Mammalian reoviruses display serotype-specific neurotropism in newborn mice, with serotype 1 (T1) strains causing hydrocephalus and serotype 3 (T3) strains causing encephalitis. However, the basis for serotype-specific disease is not clear. All reovirus serotypes bind the same proteinaceous receptors, but T1 and T3 strains engage different glycans, suggesting a function for glycan binding in serotype-specific disease. Prior to these studies, the specific glycan bound by T1 reovirus and function of glycan engagement in T1 reovirus tropism were not known. Using glycan array technology, we identified GM2 as a receptor for T1 reovirus. I demonstrated that preincubation with GM2 specifically inhibits infection of mouse embryonic fibroblasts (MEFs) by T1 but not T3 reovirus, demonstrating that GM2 is a serotype-specific receptor. Crystallography studies performed in parallel showed that the head domain of the T1 attachment protein σ1 interacts with the GM2 glycan. I used structure-guided mutagenesis and reverse genetics to engineer mutant viruses incapable of binding GM2. Mutants deficient in GM2-binding displayed impaired hemagglutination capacity and infectivity in MEFs compared with wildtype virus. A mutant T1 reovirus strain incapable of binding GM2 induced substantially less hydrocephalus than wildtype virus, an effect phenocopied by wildtype virus infection of GM2-deficient mice. These findings enhance an understanding of reovirus-glycan interactions and illuminate the function of glycan engagement in virus-induced hydrocephalus. Moreover, since reovirus is being developed as an oncolytic and vaccine vector, understanding virus-glycan interactions may enhance reovirus targeting for clinical applications.

Coronavirus (CoV) infections cause a number of human diseases ranging in severity from the common cold to severe acute respiratory syndrome (SARS). Middle East respiratory syndrome CoV (MERS-CoV) and severe acute respiratory syndrome CoV (SARS-CoV) demonstrate the continued capacity of zoonotic CoVs to cause severe human respiratory disease. The likelihood of future zoonotic CoV emergence coupled with a lack of effective therapeutics or vaccines emphasizes the need to understand mechanisms of CoV replication. CoVs have a positive-sense RNA genome that is translated into polyproteins that are processed by up to three encoded proteases. The chymotrypsin-like cysteine protease (nsp5, 3CLpro) is responsible for 11 distinct cleavages within the polyprotein. Nsp5 is required for coronavirus replication; thus, it is a key target for structure function studies and for viral inhibition. We propose a series of experiments designed to elucidate the mechanism of nsp5 activity in order to inform de novo inhibitor design. Data obtained from this study will (i) define the role of the novel third domain and interdomain loop of nsp5, which connects the catalytic fold to the third domain, during substrate selection and catalytic activity, and (ii) develop novel inhibitors of nsp5 that exploit the mechanism and functional targets of nsp5.

Coronaviruses (CoVs) are positive-strand RNA viruses that induce modifications to host-cell cytoplasmic membranes during formation of replication complexes. While important for viral replication, the dynamics of this process remain poorly understood. Existing reagents largely limited the study of these processes to fixed times, so I developed reporter viruses containing green fluorescent protein or firefly luciferase as a fusion with nonstructural protein (nsp) 2 or 3 to quantitate replication complex formation and virus replication in real time. These viruses replicated with kinetics similar to WT, demonstrating that the CoV genome has the flexibility to accommodate foreign gene addition, despite being the largest replicating RNA molecule. Use of these viruses in live-cell imaging experiments revealed continuous plasma membrane ruffling, vesicle internalization, and production of extended filopodia during CoV infection. Using several complementary techniques, I demonstrate that these membrane rearrangements are due to continuous macropinocytosis induced late during SARS-CoV and MHV infection. Additionally, I discovered that the presence of fusogenic spike on the cell surface was required for macropinocytosis induction, and that this was dependent upon EGFR activation. Ultimately, these results are the first to demonstrate the use of macropinocytosis independent from virus entry, and suggest that CoVs could exploit macropinocytosis to facilitate cell-to-cell spread during infection. During this work, a new CoV, Middle East Respiratory Syndrome (MERS) CoV, emerged in Saudi Arabia. Due to our interest in replication complex formation and membrane rearrangements, we developed a panel of antibodies to recognize MERS-CoV nsps. Several of these antibodies were highly specific for MERS-CoV nsps, allowing us to investigate the spatial and temporal formation of replication complexes in relation to cellular markers. This work is the first to visualize formation of these complexes during MERS-CoV infection and provides new reagents critical for future studies. Overall, my work has used novel reporter viruses to demonstrate the flexibility of the CoV genome, quantitate replication complex formation during infection, and provide early measurement of viral translation. Furthermore, I identified a novel function for viral exploitation of macropinocytosis and defined new virus-host interactions required for efficient CoV replication through the potential interaction of spike and EGFR.

Staphylococcus lugdunensis is a coagulase negative staphylococcus (CoNS) that is part of the normal skin flora but unlike most CoNS S. lugdunensis has the capacity to cause aggressive and rapidly progressive infections. The molecular mechanisms that facilitate the transition of S. lugdunensis from a common commensal to an invasive pathogen remain poorly defined. Analysis of the S. lugdunensis genome indicates the presence of an iron-regulated surface determinant (Isd) system with unique features. For example, unlike the Staphylococcus aureus Isd system which encodes two paralogous heme oxygenases the Isd system within S. lugdunensis encodes a single heme oxygenase. Furthermore, the terminal gene within the S. lugdunensis Isd system encodes for a predicted peptidoglycan (PG) hydrolase. My dissertation research establishes that the S. lugdunensis Isd system is iron-regulated and includes both an IsdG-family heme oxygenase and peptidoglycan hydrolase. Additionally, I have demonstrated that S. lugdunensis IsdG degrades heme to staphylobilin designating it as the conserved IsdG-mediated heme degradation product. My research also established that the peptidoglycan hydrolase within the S. lugdunensis Isd system hydrolyzes PG, modulates the release of IsdC from the PG wall and facilitates biofilm formation. Together this work provides insight into how S. lugdunensis fulfills its nutritional requirements during infection as well as how S. lugdunensis forms biofilms.

Coronaviruses are positive-sense RNA viruses that cause significant diseases in humans. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) caused a pandemic in 2002-2003, and Middle East Respiratory Syndrome coronavirus (MERS-CoV) is currently circulating in Saudi Arabia, causing severe respiratory infection with high mortality. Murine hepatitis virus is a well-established model system to study coronavirus replication. Coronavirus replication occurs in the host cell cytoplasm on cell membranes. Coronaviruses induce many membrane modifications, including double membrane vesicles (DMVs) and convoluted membranes. Three coronavirus replicase nonstructural proteins - nsp3, nsp4, and nsp6 - are required for modification of host membranes. The specific mechanisms and determinants by which these proteins interact to modify membranes are unknown. This study uses genetic approaches to introduce designed mutations in nsp3 and nsp4 in order to determine the effect during virus infection. The results of the experiments identify amino acid residues in nsp3 and nsp4 that are important for viral replication, DMV formation, and virus fitness. The data further demonstrate that intact wildtype DMV formation or morphology is not required for virus viability, but likely serves purposes in the timing and efficiency of replication. Thus nsp3-6 and the modified membranes may be novel targets for inhibition of virus replication.

Celiac disease is an immune-mediated intestinal disorder that occurs in genetically predisposed individuals exposed to dietary gluten. Viral infections are associated with the induction of celiac disease. However, not all viruses confer disease susceptibility, and little is known about specific viral characteristics that dictate immunopathologic outcomes. Feeding mice ovalbumin (OVA) as a model antigen results in systemic tolerance to OVA, which is marked by induction of regulatory T cells (Tregs) and absence of OVA-specific inflammatory T (TH1) cells. Peroral inoculation with reovirus strain T1L abrogates oral tolerance to OVA, as evidenced by a reduction in Tregs and promotion of OVA-specific TH1 cells, whereas inoculation with strain T3D-RV does not. Relative to infection with T3D-RV, T1L infection is associated with increased levels of inflammatory cytokines including type 1 interferons and interferon regulatory factor-1, which are required for virus-induced tolerance blockade. Compared to T3D-RV, T1L induces less activation of caspase-3, less sloughing of intestinal villi, and prolonged infection. To examine the relationship between apoptosis and infection in the intestine, a panel of T1L x T3D-RV reassortant viruses was recovered. The reassortant viruses capable of suppressing apoptosis in the intestine also displayed enhanced infection capacity, similar to T1L. These findings suggest that viral strains inducing minimal levels of apoptosis are capable of producing high titers of virus, which in turn may be required to stimulate the inflammatory signals required to break immunological tolerance to food antigens.

Epidemiologic studies have provided conflicting data regarding an association between Helicobacter pylori infection and iron deficiency anemia (IDA) in humans. In this thesis, I describe the use of a Mongolian gerbil model to investigate potential roles of H. pylori infection and diet in IDA. The experimental results indicate that H. pylori infection can cause IDA and that the composition of the diet influences the incidence and severity of H. pylori-induced IDA. Additionally, a high salt diet increased the incidence of gastric ulceration in infected gerbils. Finally, I examined H. pylori genetic adaptation in the Mongolian gerbil stomach. This environment is of particular interest because H. pylori-infected gerbils develop severe gastric inflammation and often develop gastric adenocarcinoma or gastric ulceration. I analyzed the whole genome sequences of H. pylori strains cultured from experimentally infected gerbils in comparison to the genome sequence of the input strain, and identified numerous polymorphisms that were positively selected in vivo.

Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode many innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV non-structural protein 14 (nsp14) is a multifunctional protein that encodes 3â-to-5â exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) activities. CoV ExoN activity performs a proofreading function and is required for high-fidelity replication of the uniquely large CoV RNA genome. Furthermore, eukaryotic mRNAs possess a methylated 5â-guanosine cap that is required for RNA stability, efficient translation, and protection from cell-intrinsic defenses. CoV nsp14 N7-MTase activity is implicated in viral RNA 5â capping resulting in structures that mimic cellular 5â caps. In this dissertation research, I tested the hypothesis that nsp14 ExoN and N7-MTase activities function to antagonize the innate immune response. Using site directed mutagenesis to ablate CoV nsp14 ExoN activity, I demonstrate that ExoN activity is required for resistance to the innate immune response and that viruses lacking ExoN activity that were generated during an antiviral state are less viable to establish a subsequent infection. In addition, I demonstrate that CoV nsp14 N7-MTase activity prevents detection of viral RNAs by innate sensors, confers resistance to the IFN-β-mediated innate immune response, and directs efficient translation of viral proteins. Altogether, this dissertation research demonstrates that CoV nsp14 ExoN and N7-MTase activities are required for viral resistance to the effects of the IFN-β-mediated innate immune response and represent highly conserved targets for the attenuation of CoVs.