PhD, University of Cincinnati, 2023, Medicine: Molecular Genetics, Biochemistry, & Microbiology

Human cytomegalovirus (HCMV) is a ß-herpesvirus that is ubiquitous in the human population. While HCMV infection generally causes mild cold- or flu-like symptoms in the immunocompetent population, people who are immunocompromised, such as newborns, are more susceptible to developing disease(s). Congenital HCMV infection is the most common congenital infection in the United States, and it is the leading viral cause of neurodevelopmental delay and the leading non-genetic cause of sensorineural hearing loss in children. Understanding more about HCMV replication and pathogenesis could pave the way for finding new treatments or a vaccine against HCMV. As a herpesvirus, HCMV can go through multiple stages of infection within a host. Initial infection and lytic replication generally occur in the epithelium of the nasopharyngeal tract, as the main transmission route of HCMV is through saliva. Once infected, these cells will begin to produce infectious viral particles that can gain access to cell types that will ultimately harbor the virus in a latent state, such as myeloid cells and hematopoietic progenitors. Infected myeloid cells can travel through-out the host's body via the bloodstream and cause secondary viremia in peripheral organs, such as the spleen, liver, kidneys, and placenta. The virus can also gain access to the bone marrow, where it can infect hematopoietic progenitor cells; this is how the virus is able to establish latency within its host. The latent infection can then be followed by multiple rounds of lytic reactivation throughout the host's lifetime. HCMV has the largest genome of all known human viruses, and thus encodes many different proteins. There are four proteins encoded in the HCMV genome that are homologous to human G protein coupled receptors (GPCRs). These viral-encoded GPCRs (vGPCRs) are UL33, UL78, US27, and US28. Though much is known about US28, very little is known about the other three vGPCRs. Thus, this thesis has focused o (open full item for complete abstract)

Committee: William Miller Ph.D. (Committee Chair); Zalfa Abdel-Malek Ph.D. (Committee Member); Stephen Waggoner Ph.D. (Committee Member); Katherine Vest Ph.D. (Committee Member); Anil Menon Ph.D. (Committee Member) Subjects: Cellular Biology

Doctor of Philosophy, Case Western Reserve University, 2023, Molecular Medicine

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus residing latently in a majority of the population. While generally asymptomatic in healthy individuals, primary infection or reactivation of latent infection in the immunocompromised, immunosuppressed, and immunonaive can lead to end organ failure if left untreated. HCMV encodes four G protein-coupled receptors (GPCRs), which play roles in both lytic and latent infection by manipulating the host cell environment, in some cases through altering cell signaling. The objective of these studies was to gain further insight into the role the HCMV-encoded GPCRs, US28 and UL33, play in the molecular mechanisms of lytic and latent infection, and in turn viral dissemination and transmission. In the first study, we demonstrate a new mechanism by which US28 controls HCMV latency. Our lab previously showed US28 is necessary for the establishment and maintenance of HCMV latency. To gain a more complete understanding of the cellular factors US28 manipulates during latency, we evaluated cellular transcriptomic changes in response to US28 expression and found this viral GPCR upregulates the cellular gene, myeloid nuclear differentiation antigen (MNDA). Previous findings revealed MNDA increases DNA-binding of the cellular transcription factor, Yin Yang 1 (YY1), which is a repressor of the major immediate early (MIE) enhancer/promoter, a region whose regulation of immediate early gene expression, in part, controls the balance between HCMV latency and reactivation. We show US28 upregulates MNDA, leading to a US28-dependent interaction between MNDA and YY1. This complex binds the MIE enhancer/promoter, resulting in transcriptional silencing of this locus, thereby aiding in the maintenance of latent infection. These studies reveal a new mechanism by which US28 regulates the MIE enhancer, through the control of cellular proteins MNDA and YY1. Secondly, we evaluated the HCMV-encoded GPCR UL33 and its role in viral replication. P (open full item for complete abstract)

Committee: Christine O'Connor, Ph.D. (Advisor); Robert Silverman, Ph.D. (Committee Chair); Michelle Longworth, Ph.D. (Committee Member); Robert Fairchild, Ph.D. (Committee Member); Christine Koval,, M.D. (Committee Member) Subjects: Biology; Microbiology; Virology

PhD, University of Cincinnati, 2022, Medicine: Molecular Genetics, Biochemistry, & Microbiology

Human cytomegalovirus (HCMV) is a highly successful pathogen which is prevalent throughout the world and estimated to infect up to 80% of the human population globally. HCMV causes lifelong persistent infections which can severely impact the health of those who are immunocompromised and lead to increased risk of developing other diseases for even the immunocompetent. Epithelial cells make up numerous tissues and organ systems throughout the body and represent a biologically important cell type for HCMV infection; however, epithelial models are limited and HCMV pathogenesis is most extensively studied in the context of retinal epithelial infection. The salivary glands are an important site for HCMV replication as the virus establishes a persistent infection in acinar or ductal epithelial cells which then transmit infectious virus to new hosts via saliva. This contrasts with other tissue systems which are marked by an acute infection, followed by clearance of the virus through host immune system function. We explored HCMV epithelial tropism in these studies by use of a primary human salivary gland-derived epithelial (hSGE) cell system. During infection of hSGE cells we observed that, in contrast to the established epithelial model, HCMV can persistently infect these cells by use of self-limiting lytic replication. This self-limiting of infection is likely one of many mechanisms used by the virus to avoid immune detection and subsequent clearance. We additionally identified that epithelial defective strains of HCMV can be greatly enhanced for epithelial infection in hSGE and other epithelial models by use of histone deacetylase inhibitors. These inhibitors were clearly identified to be capable of overcoming the loss of the Pentamer, which is an essential viral protein for replication in epithelial cells. This Pentamer is described as being an entry mediator by unclear mechanisms. Our data reveals that virions lacking Pentamer can be rescued post-entry and recontextuali (open full item for complete abstract)

Committee: William Miller Ph.D. (Committee Member); James Bridges (Committee Member); David Plas Ph.D. (Committee Member); Anil Menon Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member) Subjects: Virology

PhD, University of Cincinnati, 2020, Medicine: Pathobiology and Molecular Medicine

While cytomegalovirus (CMV) infection poses a considerable burden to public health, an efficacious vaccine is not yet available. Classical vaccine efforts focusing on the induction of Band T-cell memory have proven ineffective and, therefore, CMV remains a significant public health threat. Notably, CMV infections in both mice and humans trigger accumulation of unique adaptive natural killer (NK) cell subsets that putatively possess enhanced effector functions against CMV. This suggests that a pioneering approach to creating an efficacious CMV vaccine would include stimulation of the accumulation and long-term maintenance of these CMVreactive NK cells. It remains unknown, however, whether current CMV vaccines can stimulate the expansion of these NK-cell subsets. Here, we interrogated this question using a unique set of samples collected during a CMV vaccine trial. Vaccine study participants did not show an expansion of the CMV-dependent NKG2C+ NK cell subset, but marked oscillations of the frequency of FcR?neg NK cells uniformly lacking FcR?, EAT-2 and SYK adaptor molecules. Interestingly, the FcR?neg NK cells in this healthy cohort were phenotypically and functionally distinct from the ones reported in CMV-infected individuals. This unique longitudinal data in healthy CMV-seronegative individuals indicates that NK-cell subsets may react to unknown environmental or inflammatory cues and demonstrate greater flux than appreciated from past cross-sectional studies. We further evaluated the functional capabilities of CMV-associated NK subsets present in healthy individuals by using peripheral blood mononuclear cells (PBMC) from healthy blood donors. Since NKG2C+ and FcR?neg NK subsets are present in minute quantities in the blood of healthy individuals, we used a K562 feeder cell line with membrane bound IL-21 in order to expand those ex vivo for subsequent functional interrogation. Within the limit of our small sample size we report no significant d (open full item for complete abstract)

Committee: Stephen Waggoner Ph.D. (Committee Chair); David Bernstein M.D. (Committee Member); Jason Blackard Ph.D. (Committee Member); Laura Conforti Ph.D. (Committee Member); Rajat Madan M.D. Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy (PhD), Ohio University, 2018, Molecular and Cellular Biology (Arts and Sciences)

Human cytomegalovirus (HCMV) is primarily an opportunistic pathogen in human, causing significant disease in immunocompromised individuals. A large, double-stranded DNA genome (~230 kilobases) provides the coding capacity for over 200 genes, of which only 25% are required for viral replication in cell culture. The viral UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are essential for replication, and viruses lacking the proper expression of pUL34 cannot replicate in cell culture. Interactions of pUL34 with DNA binding sites (US3 and US9?) represses transcription of (these) two viral immune evasion genes that are dispensable for replication in cell culture. There are 12 additional predicted pUL34-binding sites present in the HCMV genome (strain AD169), with three of them concentrated near the HCMV origin of lytic replication (oriLyt). Analysis of 47 clinical isolates of HCMV confirmed that the predicted UL34-binding sites were highly conserved. Protein-DNA interactions were analyzed during infection with ChIP-seq and confirmed that pUL34 binds to the human and viral genome during infection, including at the three predicted UL34-binding sites in the oriLyt region. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated, oriLyt-dependent DNA replication. Subsequently, mutagenesis of these same sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with 3 virus proteins that are essential for viral DNA replication - IE2, UL44, and UL84, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Lastly, mutagenesis of the predicted UL34-binding site in the third exon of another essential viral gene, UL37, demonstrated that some UL34-binding sites are not important for viral replication.

Committee: Bonita Biegalke (Advisor); Calvin James (Committee Member); Mark Berryman (Committee Member); Justin Holub (Committee Chair) Subjects: Genetics; Molecular Biology; Virology

MS, University of Cincinnati, 2016, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Human Cytomegalovirus (HCMV), also known as human herpesvirus 5 (HHV5), and the related rodent and non-human primate cytomegaloviruses (MCMV, RCMV and rhCMV), encode G-protein coupled receptors (GPCRs) that appear to play important roles during CMV infection including viral dissemination, cellular migration, and viral latency. The rodent viruses encode two GPCRs, while the human and non-human primate viruses encode four GPCRs. Although divergent in primary amino acid sequence, these viral encoded GPCRs share highest homology with members of the chemokine receptor (CKR) subfamily including CCR1 and CCR5. The HCMV encoded GPCR, US28 is particularly interesting due to its ability to exploit multiple G-protein dependent and G-protein independent pathways. One signaling pathway that has been shown to be US28-dependent but independent of ligand activation is the Gaq/11 mediated phospholipase Cß (PLCß) pathway. PLCß hydrolyzes phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The production of DAG and IP3 promotes the downstream activation of protein kinase C (PKC) and the release of Ca2+ from intracellular stores, respectively. Several studies have been conducted which indicate important roles for US28 during viral infection, however, the full extent of US28 signaling pathways and how these signaling pathways contribute to the US28 function during infection has yet to be uncovered. Furthermore, the predominant models used to study CMV infection and viral GPCR signaling are that of mice and immortalized human cell lines. Studies in mice have revealed essential roles for the MCMV GPCRs in facilitating viral replication in salivary tissues in vivo which is important for horizontal transmission, however similar studies with the HCMV GPCRs have not been carried out due to limitations in tissue and cellular systems. With recent advances in various tissue engineering strategies, it has now become possible to cult (open full item for complete abstract)

Committee: William Miller Ph.D. (Committee Chair); Edmund Choi Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member) Subjects: Molecular Biology

PhD, University of Cincinnati, 2015, Medicine: Molecular Genetics, Biochemistry, and Microbiology

Human cytomegalovirus (HCMV) is a member of the subfamily of Beta-herpesviridae that establishes latency in human hematopoietic progenitor cells, myeloid progenitor cells and monocytes. While the serological positive population is prevalent worldwide, this virus does not cause severe symptoms in immunocompetent individuals. However, in immunocompromised patients and developing fetuses, this virus can cause serious morbidity and mortality. In light of many chronic diseases in the modern world leading to immunodeficiency and organ transplants, the importance of HCMV in medical research and public health is rising. Although it is known that HCMV establishes latency after primary infection, the molecular mechanism(s) that controls latent/lytic switch remains elusive. In addition, there is limited knowledge about stimuli that could induce the myeloid cells to support HCMV lytic phase. In this thesis, we studied the effect of vitamin D, a hormone produced by human body or acquired from diet, on HCMV replication in peripheral monocytes and THP-1 cells. We found that vitamin D induces monocyte differentiation to facilitate HCMV replication, and instead of causing a direct up-regulation of HCMV immediate-early gene promoter (MIEP) activity as phorbol 12-myristate 13-acetate (PMA) treatment, the vitamin D induced monocyte differentiation causes an open chromatin structure around MIEP enhancer region of HCMV genome which also happens in PMA treated monocytes. In Chapter III of the thesis, we examined the Gαq dependent signaling pathways triggered by herpesviruses encoded G protein-coupled receptors by using Gαq specific inhibitor (YM-254890). We found that the inositol triphosphate accumulation triggered by US28 and M33 can be blocked by the inhibitor but the inositol triphosphate accumulation initiated by ORF74 cannot be suppressed by the inhibitor. In addition, CREB and NFAT activated by US28 and M33 can also be inhibited by the inhibitor, showing that they are Gαq depend (open full item for complete abstract)

Committee: William Miller Ph.D. (Committee Chair); Rhett Kovall Ph.D. (Committee Member); Carolyn Lutzko Ph.D. (Committee Member); Simon Newman Ph.D. (Committee Member); Richard Thompson Ph.D. (Committee Member) Subjects: Virology

PhD, University of Cincinnati, 2014, Medicine: Molecular Genetics, Biochemistry, and Microbiology

The betaherpesvirus Human Cytomegalovirus (HCMV) is estimated to be present in 40-80% of world population. HCMV infection in a healthy person causes mild symptoms, although the virus persists in the host in the latent form. Immunocompromised patients such as HIV and organ transplant patients as well as fetuses and neonatal babies with underdeveloped immune system are most affected by HCMV infection. CMVs are characterized by their strict species specifity; therefore humans are the only host for HCMV. The mouse cytomegalovirus (MCMV) has been the most useful animal model to explore HCMV spread and disease. CMVs encode G Protein-Coupled Receptors (GPCR) and these viral GPCRs are increasingly recognized as important regulators of pathogenesis and disease. HCMV for example encodes four of these GPCRs, whereas MCMV expresses two: M33 and M78. M33 shows constitutive signaling properties and activates various signaling pathways. M33 stimulates the PLC-&beta/PKC pathway via G&alphaq/11, however activation of NF-&kappaB and p38 MAP-kinase is independent of G-proteins. Importantly, M33 is essential for dissemination to or growth in salivary glands of immunocompetent mice and the G-protein signaling ability of M33 is necessary for viral growth at this site. Here we used the non-obese diabetic (NOD) and the immunocompromised NOD scid gamma (NSG) mouse models to assess a potential role for M33 as an immunomodulator and to further investigate the mechanism(s) by which M33 promotes viral growth in vivo. We are able to detect replication of &DeltaM33 viruses in the salivary glands of immunocompromised NSG mice; however it exhibited a 400x defect compared to wildtype MCMV. Since the growth phenotype is not completely reverted in the NSG mice, we conclude that M33 is not solely functioning to modulate the immune system. We also showed that M33 is dispensable for hematogenous dissemination within the host and that activation of G&alphaq signaling pathways alone is not sufficient to pr (open full item for complete abstract)

Committee: William Miller Ph.D. (Committee Chair); Nancy Sawtell Ph.D. (Committee Member); Kasper Hoebe Ph.D. (Committee Member); Richard Thompson Ph.D. (Committee Member); Alison Weiss Ph.D. (Committee Member) Subjects: Virology

PhD, University of Cincinnati, 2009, Medicine : Molecular Genetics, Biochemistry, and Microbiology

The human Cytomegalovirus (HCMV) is a species specific herpes virus which encodes several G-protein coupled receptors (GPCRs) that likely serve important roles in immunomodulation and viral dissemination during infection. One of these receptors, called US28, has been shown to act through multiple G-protein dependent and G-protein independent signaling pathways, which contribute to diverse US28-mediated effects following HCMV infection such as cell migration and tumorigenesis. One of these signaling pathways occurring in HCMV infected cells, the Gαq/11 mediated PLC-β pathway, has previously been shown to be dependent on US28, but does not necessarily require ligand activation by chemokines. Additionally, these signaling events are likely attenuated or “desensitized” in a sequence of events including receptor phosphorylation and β-arrestin recruitment to the carboxy terminus of the receptor. The full repertoire of signaling events activated by US28, their dependence on chemokine binding, and attenuation by cellular regulatory factors is still unknown. Most studies regarding US28 activity have been performed by over-expressing the receptor or various mutants in cell culture in the absence of the other 200 gene products encoded by HCMV, and with expression levels of US28 which may not be physiologically relevant. To address this, recombinant HCMVs knocked-out for US28 or encoding FLAG-tagged versions of either wild type US28, a signaling deficient DRY box mutant US28(R129A), a chemokine binding mutant US28ΔΝ a carboxy terminal mutant US28(1-314) were engineered into the HCMV clinical isolate VR1814 via BAC recombineering. These viruses were first used to verify that US28 primarily exhibits early kinetics, with expression peaking at 48-72 hours post infection, although very low levels can be detected as early as 6 hpi. We then verified that the FLAG-tagged US28 wild type has the same ability to induce PLC-beta signaling potential as the parental HCMV strain, indicating t (open full item for complete abstract)

Committee: William Miller PhD (Committee Chair); Gary Dean PhD (Committee Member); Daniel Hassett PhD (Committee Member); JoEl Schultz PhD (Committee Member); Richard Thompson PhD (Committee Member); Steve Liggett PhD (Committee Member) Subjects: Biochemistry; Biomedical Research; Molecular Biology; Virology

PhD, University of Cincinnati, 2008, Medicine : Molecular Genetics, Biochemistry, and Microbiology

Several members of the herpesvirus family encode seven transmembrane-spanning proteins that share significant homology with the cellular G protein-coupled receptors (GPCRs). These viral GPCR homologs stimulate both G protein-dependent and G protein-independent signaling pathways reminiscent of cellular GPCRs. By mimicking host cell proteins and altering intricate signaling networks, viral GPCRs are often viewed as immune evasion genes, enhancing viral replication and persistence within the infected host. The studies described herein characterize the signaling activities and regulation of the viral GPCR homolog M33 encoded by the murine cytomegalovirus (MCMV). Cytomegaloviruses are species-specific opportunistic pathogens that cause disease within the immunocompromised host. MCMV has been used extensively as a model for studying human cytomegalovirus (HCMV) pathogenesis in vivo, with M33 playing an essential role in MCMV dissemination and replication within the host salivary gland. We have identified the first ten amino acids of M33 as critical for proper M33 post-translational modification, cell surface expression, as well as the activation of G protein signaling events. We show through mutational analysis that a conserved GPCR signaling motif of M33 is required for G protein signaling yet is not involved in other M33 signaling activities including phosphorylation of the mitogen-activated protein kinase p38 and activation of the transcription factor NF-κB, indicating M33 possesses G protein-independent signaling abilities. Additionally, we have identified a role of cellular GPCR regulatory proteins in attenuating M33 G protein signaling through a dual mechanism involving both receptor phosphorylation and sequestration of activated G proteins. Lastly, in order to understand M33 signaling and regulation in the context of MCMV infection we made various mutations of the M33 ORF within the MCMV genome and show that M33 and its ability to activate a Gq/11/PLC-β/PKC pathwa (open full item for complete abstract)

Committee: William Miller PhD (Committee Chair); Rhonda Cardin PhD (Committee Member); Gary Dean PhD (Committee Member); Rhett Kovall PhD (Committee Member); Alison Weiss PhD (Committee Member) Subjects: Biochemistry; Microbiology; Molecular Biology; Virology

MS, University of Cincinnati, 2007, Medicine : Epidemiology (Environmental Health)

Background: Cytomegalovirus (CMV) is one of the leading causes of birth defects and childhood disability in the US. Methods: To determine the CMV seroprevalence of adolescent females, serum antibody in 12-17 year olds at Cincinnati Children's, UT Medical Branch at Galveston, and Vanderbilt University were studied. To determine risk factors associated with CMV (particularly sexual activity) a comprehensive questionnaire was administered. Results: The seroprevalence of CMV was 52.2%. Both sexual activity and children in the home were statistically significant risk factors with an approximate two fold increased risk in CMV antibody. Being of white race had an inverse relationship with CMV. Conclusion: Identification of CMV negative females is important to develop methods of preventing acquisition of CMV during conception and childbearing years. Adolescent females who have children in the home and are sexually active likely have already been infected with CMV. A larger sample size may indicate additional associated risks.

Committee: Dr. Kim Dietrich (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2008, Molecular, Cellular, and Developmental Biology

Human cytomegalovirus (HCMV) is a ubiquitous pathogen infecting 60- 100% of the world population. In populations that are immunosuppressed including AIDS patients, transplant recipients, and neonates who have immature immune systems this virus causes severe disease and morbidity. After primary infection of the host via contact with contaminated body fluids the virus persists in the host and establishes latency. The virus reactivates from latency multiple times and is shed in the host body fluids. The ability of HCMV to establish such lifelong infections is attributed in part to its ability to modulate various aspects of host immune responses.Central to the antiviral immune response of the host is interferon (IFN) mediated activation of cellular gene products that interfere with various aspects of the viral life cycle. It is known that IFN mediated signaling is attenuated in HCMV infected cells. We set out to identify the HCMV gene products that are involved in modulating IFN signaling and elucidate their mode of action. For this purpose we constructed a cDNA library of HCMV laboratory strain AD169. Characterization of this cDNA library revealed an abundance of non protein coding transcripts and transcripts in antisense orientation to known or predicted HCMV genes. We screened this cDNA library for genes that disrupt IFN signaling, and the UL123 gene encoding the IE1 protein was the first candidate gene identified. Our results indicate that IE1 can attenuate both type I and type II IFN signaling. We found that IE1 does not inhibit the proximal events of type II IFN signaling but it interferes with STAT1 binding to target DNA elements. Using a series of truncation mutants we were able to map this function to the C terminal domain of the IE1 protein. Our data indicate that a novel mechanism is employed by IE1 to interfere with the association of STAT1 with its target DNA. The research findings presented here reveal the increased complexity of the HCMV transcriptome an (open full item for complete abstract)

Committee: Joanne Trgovcich PhD (Advisor); James Waldman PhD (Committee Member); Marshall Williams PhD (Committee Member); Daniel Sedmak MD (Committee Member) Subjects: Molecular Biology; Virology

Doctor of Philosophy, The Ohio State University, 2007, Integrated Biomedical Science

Human cytomegalovirus (HCMV) is a beta-herpes virus which can cause serious disease and even death in congenitally-infected infants and in immunocompromised individuals or immunosuppressed transplant recipients. Sphingolipids are structural components of cell membranes that can act as critical mediators of cell signaling. An unexplored area of research is whether HCMV modulates sphingolipids and their signaling pathways. Our data show that HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK) within different cell types and occurs during early times of infection. Measuring the levels of transcripts encoding key enzymes of the sphingolipid metabolic pathway during HCMV infection revealed a temporal regulation of both synthetic and degradative enzymes of this pathway. Using mass spectrometry, we were able to generate a sphingolipidomic profile of HCMV-infected cells, which suggests an enhancement of de novo sphingolipid synthesis at early times of infection. This was followed by a decrease in the levels of several sphingolipids at 48 hrs, correlating with the upregulation of degradative enzymes. Then by knocking down SphK1 expression with siRNA, we showed that this enzyme may function within HCMV infected cells to sustain levels of the immediate early (IE) transactivator, IE1. Later, we show evidence suggesting that de novo sphingolipid biosynthesis is necessary for the production of optimal levels of infectious virus progeny, but has an intermediate product which suppresses the levels of IE1 protein accumulation. Through exogenous addition of dhSph, an intermediate of de novo sphingolipid synthesis, we show that this lipid can inhibit HCMV protein accumulation. dhS1P treatment, however, results in increased accumulation of HCMV proteins, although only at early times during infection. Moreover, pretreatment of cells with dhS1P or S1P prior to infection results in reduced accumulation of HCMV gene products, thus indicating a time-de (open full item for complete abstract)

Committee: James Van Brocklyn (Advisor) Subjects:

Doctor of Philosophy, The Ohio State University, 2005, Pathology

Leflunomide is an experimental immunosuppressive agent that has shown efficacy as an antiviral agent against human cytomegalovirus (HCMV) and polyomavirus strain BK (BKV). An antiviral regimen has been approved for immunosuppressed patients suffering complications from HCMV infection, whereas a good treatment option for patients infected with BKV does not exist. Unfortunately, the antiviral treatment options for patients infected with HCMV have helped promote the propagation of multi-drug resistant HCMV strains. This body of work illustrates the possible antiviral mechanisms associated with Leflunomide using an in vitro model system. We have tested the hypothesis that the antiviral activity of A77 1726, the active metabolite of Leflunomide, is a result of its inhibition of phosphorylation of one or more viral structural proteins. Western blot, Southern (Dot Blot) blot, and CMV gene array analysis demonstrated that Leflunomide does not inhibit HCMV DNA synthesis, the translation of essential viral proteins, or the transcription of viral mRNA. 32P-orthophosphate labeling experiments confirm a reduction in the phosphorylation of more than one of the HCMV tegument proteins. In addition, immunohistochemical staining showed discrete changes in localization of these tegument proteins in Leflunomide-treated cells. Co-immunoprecipitation experiments confirm that Leflunomide disrupts the interaction of viral tegument proteins suggesting that Leflunomide may inhibit complete infectious virion assembly by altering the phosphorylation states of one or more viral structural proteins. The second aspect of this work was to determine if Leflunomide would inhibit the replication of BKV, a polyoma virus unrelated to HCMV. We tested the hypothesis that A77 1726 would inhibit the production of infectious BKV particles without inhibiting DNA synthesis or large T antigen translation. Plaque assay data demonstrated a log decrease in viral titers when infected cells were treated with A77 17 (open full item for complete abstract)

Committee: W. Waldman (Advisor) Subjects: