David J. Miller, BSc, PhD - US grants

Our long-range objective is to develop a clearer fundamental understanding of the molecular basis of fertilization so that the process can be controlled to either promote fertilization or block it. Much progress has been made to identify ZP3, the protein in the mammalian egg coat that binds sperm and stimulates the acrosome reaction, the release of the acrosomal vesicle that must occur for sperm to penetrate the egg coat. Despite this progress, the identity of the ZP3 receptor on sperm has been elusive. One strong candidate is beta1,4-galactosyltransferase, an enzyme first discovered in the Golgi apparatus of somatic cells but later found on the surface of sperm. Beta1,4-Galactosyltransferase binds ZP3 but not other egg coat proteins. In addition to its role in gamete adhesion, beta1,4- galactosyltransferase acts to trigger the acrosome reaction in sperm. The acrosome reaction can be stimulated with antibodies to beta1,4-galactosyltransferase that act as mimics of ZP3 to bind and crosslink beta1,4-galactosyltransferase. There is evidence that binding beta1,4-galactosyltransferase stimulates at least some of the same signaling systems inside sperm that ZP3 stimulates, leading to the proposal that beta1,4- galactosyltransferase may be the major ZP3 receptor signaling the acrosome reaction. Mice with a targeted deletion of the beta1,4-galactosyltransferase gene, although fertile, produce sperm with severely compromised ability to acrosome react and penetrate the egg coat. In this proposal, the signaling systems that are activated by beta1,4-galactosyltransferase will be studied. The goals are to determine if binding beta1,4-galactosyltransferase specifically can completely reproduce all of the identified signaling systems activated by ZP3 binding, to study beta1,4-galactosyltransferase mutants to identify structural features necessary for signaling, and to identify proteins that interact with the portion of beta1,4- galactosyltransferase found in the cytosol. These studies will clarify the role of a sperm receptor for the egg coat in the acrosome reaction, a necessary step in the process of fertilization.

The career development of physician-scientists requires a broad approach in preparation for a career in academic medicine with research, teaching, and clinical responsibilities. This grant proposes a four-year career development plan consisting primarily of targeted hypothesis-driven research, but also incorporating teaching and mentoring activities, participation in institutional and national conferences and meetings, and continued clinical training. The University of Wisconsin-Madison provides the ideal environment to develop a career as a physician-scientist, with a reputation for premiere medical care and cutting-edge biomedical research, state-of-the-art facilities, extensive technical and intellectual resources, and a strong tradition of open collaboration among researchers and clinicians. The proposed research will investigate the dynamic and complex host-pathogen interactions involved in viral replication and pathogenesis. The simple genomic structure of positive-strand RNA viruses and experimental evidence suggest that host factors are indispensable for their replication, although the identity of these host factors and their functional impact on viral replication remain largely unknown. The recent development of novel viral replication systems in the well-studied yeast Saccharomyces cerevisiae permits the functional investigation of the cellular host factors involved in positive-strand RNA virus replication. Flock house virus (FHV), a bipartite positive-strand RNA virus and member of the Nodaviridae family, is the only animal virus shown to replicate in S. cerevisiae, and consequently represents a unique model to investigate the mechanisms of viral replication. The S. cerevisiae experimental system will be used to isolate and characterize yeast mutants unable to support FHV replication. A concurrent immunochemical approach will also be used to isolate and characterize cellular host proteins associated with the FHV RNA polymerase. The identification and characterization of host-pathogen interactions, with a particular emphasis on the impact of cellular host factors, are crucial both to understand the basic mechanisms of viral replication and pathogenesis and to design rational antiviral therapies.

Viral pathogenesis is intimately linked with dynamic and complex host-pathogen interactions. The mechanisms underlying these essential interactions, in particular those shared by viruses with similar genomic structures, represent attractive potential targets for antiviral drugs. Viruses that contain a positive-sense single-stranded RNA genome, such as hepatitis C virus, West Nile virus, and the SARS coronavirus, represent a diverse group of pathogens responsible for significant human diseases for which few effective therapies exist. Despite the varied clinical syndromes caused by these viruses, all characterized positive-strand RNA viruses use intracellular membranes for viral RNA replication complex formation and function. However, the mechanisms whereby viral RNA replication complexes assemble on specific intracellular membranes are not well understood. The long-term objectives of this project are to elucidate the host-pathogen interactions that facilitate positive-strand RNA virus replication complex assembly and function. A detailed understanding of these interactions will provide insight into the mechanisms of viral pathogenesis, and potentially identify novel targets for broadly effective antiviral drugs. The specific focus of this proposal is to define the early events in viral RNA replication complex assembly. The targeting, transport, and initial interactions of virus-encoded RNA replication complex proteins with intracellular membranes are essential steps in replication complex assembly, and therefore are important determinants of viral pathogenesis. The general strategy of the proposed research is to use Flock house virus, an established and versatile model used to study positive-strand RNA virus structure and replication, to investigate the mechanisms of viral RNA replication complex assembly. Biochemical, molecular, and genetic approaches will be used to investigate the targeting and transport of the Flock house virus RNA-dependent RNA polymerase to intracellular membranes. The specific aims of this proposal are designed to accomplish two goals: 1) understand the role of cellular chaperone proteins in Flock house virus RNA replication complex assembly; and 2) define the membrane receptor responsible for Flock house virus RNA replication complex intracellular localization.

[unreadable] Description (provided by applicant): Innate cellular antiviral responses are essential first lines of defense against viral infections, and also play crucial roles in the initiation of adaptive immunity and hence eventual virus control or clearance. However, the inappropriate activation of innate immune responses can also contribute to viral pathogenesis. Intracellular pathways that suppress or potentiate steps in innate antiviral responses therefore represent crucial components of the host-pathogen interactions that ultimately determine the outcome of viral infections. Furthermore, a detailed understanding of the cellular and molecular mechanisms underlying these pathways is important for the rationale design of novel antiviral or immunomodulatory drugs. Although our knowledge regarding innate cellular antiviral responses has progressed tremendously over the past decade, significant gaps still exist. For example, innate antiviral immune responses consist of a complex network of interactions that exhibit both cell type- and pathogen-specific differences, but the functional significance and impact of these differences on viral pathogenesis, particularly within the central nervous system, have not been well studied. We hypothesize that intrinsic maturation-dependent innate antiviral responses are key determinants in the pathogenesis of neurotropic virus infections. We propose to explore these responses in human neuronal cells using western equine encephalitis virus (WEEV), a mosquito-borne positive-strand RNA virus belonging to the Togaviridae family (genus: Alphavirus). WEEV and the closely related eastern and Venezuelan equine encephalitis viruses are category B bioterrorism agents, and cause central nervous system infections that are associated with high clinical morbidity and mortality for which there are no effective therapies. Arboviral diseases, such as those caused by neurotropic alphaviruses, have also seen both an emergence and resurgence as significant public health threats over the past several decades. The long-term objectives of this project are to elucidate the molecular mechanisms underlying the cell- autonomous recognition, activation, and effector pathways involved in the innate antiviral responses of human central nervous system neurons to arbovirus infections. The focus of this proposal is to employ targeted and global genetic and chemical genomic approaches with cultured human neuronal cells to examine the activation pathways responsible for WEEV recognition and initiation of innate antiviral responses in mature neurons. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses are transmitted by mosquitoes, cause serious and potentially fatal central nervous system infections in humans, and are considered NIAID Category B Priority Pathogens due to their potential misuse as bioterrorism agents. Although vaccine development is in progress for several alphaviruses, there is an urgent and pressing need for broadly active antiviral agents against these virulent pathogens. Studies with experimental alphavirus encephalitis in mice have shown that while neurons are damaged directly by virus, uninfected neurons are also damaged via bystander mechanisms that involve altered homeostatic and neuroprotective functions of microglial cells or astrocytes. Thus, we hypothesize that combination therapy that directly targets virus replication and enhances neuroprotective responses will provide synergistic benefit in viral encephalitis. To identify and develop new antivirals to test this hypothesis, we recently screened a chemically defined small molecule library and identified a thieno[3,2-b]pyrrole compound that has potent activity against neurotropic alphaviruses in culture. Furthermore, a limited structure-activity relationship analysis with twenty structurally related analogs identified six additional compounds with enhanced in vitro activity. In addition, to explore the innovative use of natural products as a source for novel antivirals, we analyzed a series of extracts derived from marine actinomycetes and identified several that contained potent activity against alphaviruses. We are currently positioned to rapidly and efficiently move candidate antivirals through preclinical development, and we have assembled a team of experienced investigators with diverse expertise in the fields of virology, neurology, pathology, physiology, and medicinal chemistry to accomplish this task. The long-term goals of this highly collaborative project are to develop effective and broadly active therapies for encephalitis caused by neurotropic alphaviruses and related arboviruses. The specific aims of this proposal are: (1) targeted chemical modification of lead antiviral compounds, based on the initial structure-activity relationship analysis, to enhance potency, reduce toxicity, improve solubility and metabolic stability, and optimize membrane permeability;(2) identify molecular target(s) responsible for their antiviral activity;(3) analyze the in vivo pharmacokinetics and efficacy of candidate antivirals, including combination treatment with neuroprotective agents;and (4) isolate and characterize novel compounds with antiviral activity derived from marine microbes.

DESCRIPTION (provided by applicant): Mosquito-borne viruses, or arboviruses, are among the most important emerging pathogens worldwide with a significant potential impact on human health, and are also prominent components of the NIAID Category A, B, and C priority pathogens lists. The inclusion of many arboviruses as high priority pathogens is due in part to their virulence, the potential for vector-mediated dissemination, and the public concern regarding insect-borne viral infections. In addition, despite the worldwide impact of arboviruses, few effective treatments are currently available. The objective of this proposal is to use western equine encephalitis virus (WEEV), a category B arbovirus, to identify and develop novel antiviral compounds isolated from natural product extracts produced by marine sediment-derived microbes. Compounds derived from terrestrial and marine microorganisms have historically provided a rich source for novel therapeutics against a range of microorganisms, but have thus far been underutilized in the development of antiviral agents. We have already developed, validated, and employed a WEEV replicon-based assay to complete high-throughput screens against a library of >16,000 pre- fractionated extracts derived from a diverse array of marine microbial species. Furthermore, we have completed both analytical and preparative preliminary chromatographic fractionation procedures for two distinct extracts with validated antiviral activity. The specific aims of this proposal are designed to complete the isolation and functional characterization of active compounds from these two candidate extracts, including structural examination and initial viral target identification, and to expand the identification of microbe-derived natural products with anti-arboviral activity. The long term goals of this research project are to develop a wide range of antiviral compounds derived from marine microbes, characterize their structures and antiviral activities, and complete their preclinical testing in animal models of arbovirus-mediated disease, with the ultimate objective being clinical implementation of these candidate novel drugs. The experiments outlined in this exploratory project proposal will be used to establish the groundwork to accomplish these goals. PUBLIC HEALTH RELEVANCE: This study is designed to identify new drugs to treat central nervous system infections caused by potentially deadly viruses that are transmitted by insects. This research is important to public health because there are currently few effective medications to treat mosquito-borne viral infections, and preventative strategies such as vaccination are still in the developmental phases. In addition, routine vaccine distribution and use may be difficult to implement on a population-wide basis due to safety concerns with mass vaccination in the absence of an outbreak scenario. Therefore, the availability of effective medications to either treat established disease or prevent infection is a highly valuable component in the overall strategy to combat the potentially devastating diseases caused by these viruses.

Project Summary Fertility depends on successful fertilization and early development, processes that occur in the oviduct. Common therapies for human infertility, such as in vitro fertilization and intracytoplasmic sperm injection, are expensive and increase risks of a variety of problems. More knowledge of how the oviduct interacts with sperm, the cumulus-oocyte complex (COC) and the developing embryo may improve fertility and reduce the need for therapies. The oviduct serves as a reservoir for sperm, after semen deposition and before fertilization. Binding to the oviduct maintains sperm viability and suppresses motility. Sperm are released to move to the upper oviduct (ampulla) to fertilize oocytes. There are many gaps in this model of sperm-oviduct interaction. Our studies have begun to fill some of these gaps. We have used a glycomic approach to screen 377 glycans and found that all glycans with affinity for porcine sperm have either of two motifs, sulfated Lewis X trisaccharide or branched 6-sialylated complex glycans. We have identified two candidate receptors for both glycans on the sperm membrane, PKDREJ and ADAM5. Notably, mouse sperm deficient in PKDREJ and other ADAMs do not accumulate beyond the utero-tubal junction, but it is not known if this is due to a problem in binding and retention in the oviduct. Remarkably, if these glycans are immobilized on beads, they can extend sperm lifespan, much like binding to oviduct cells prolongs the lifespan of sperm. Finally, we found that cumulus-oocyte complexes (COCs) secrete molecules that signal sperm release from the lower oviduct so they can move toward the site of fertilization. The Specific Aims will provide a mechanistic understanding of how sperm bind the oviduct, how binding prolongs sperm lifespan and how sperm are released from the oviduct by the COC. Aim 1: To determine the function of PKDREJ and ADAM5 in sperm by blocking each protein and mutating each gene in swine. Sperm from pigs that are deficient in each of these proteins will be examined to determine if their ability to bind oviduct cells and their fertility is affected. Aim 2. To determine if sperm binding to immobilized oviduct glycans suppresses ROS production, conserves ATP, and stabilizes the plasma membrane, which extends sperm lifespan and fertility. Sperm bound to immobilized glycans will be examined to ascertain what changes adhesion induces. The second group of experiments will determine which changes are sufficient to prolong lifespan by adding agents that scavenge ROS and stabilize the plasma membrane. Aim 3. To determine how the cumulus-oocyte complex signals release of sperm from oviduct cells and glycans. We will examine whether progesterone, prostaglandins, or proteins that may be secreted from COCs might induce porcine sperm release from oviduct cells and immobilized glycans. The completion of these Specific Aims will elucidate how sperm bind to the oviduct, are stored in the oviduct and are released in response to the cumulus-oocyte complex. This fundamental information could be used to develop simpler, safer and less costly assisted reproductive technologies.