Thomas M. Gallagher - US grants

DESCRIPTION (Adapted from Applicant's Summary): The murine coronaviruses include a large collection of different strains, with each strain infecting different tissues and thus causing a unique disease pattern. Diseases include hepatitis, gastroenteritis, and chronic encephalomyelitis, the latter serving as a model for human neurodegenerative disease. Tropism of the different viruses for distinct tissues is controlled in large part by structural variations within the virion spike glycoprotein. The spike is a complex oligomeric structure that mediates virus binding to specific cellular receptors as well as fusion of viral and cellular membranes. Fulfillment of these functions delivers viral genomes into cells, thereby establishing infection and subsequent disease. Because spike is central to several essential steps in viral infection, tropism and pathogenesis, the long-term objective is to describe the molecular mechanisms by which spike proteins carry out the functions required for genome delivery. The investigator will investigate how naturally occurring variation in the coronavirus spike protein impacts the efficiency of oligomerization, incorporation into virions, and ultimate genome delivery functions. Various spikes will be synthesized from cDNA in conjunction with components required for pseudovirion assembly and virion incorporation efficiencies will be measured. The applicant will determine how the same structural variations alter the kinetics of spike binding to its cellular receptor and spike-mediated membrane fusion. Using soluble forms of the cellular receptor, he will attempt to trigger conversion of the spikes into a fusion-active conformation. Fusion conformations will be identified by liposome binding assays, and putative conformational changes will be probed using spike-specific monoclonal antibodies. The use of site-directed mutants will allow them to identify regions on the receptors that are required to stimulate the formation of fusion conformations. These findings will be used to build a model depicting various stages of coronavirus entry into cells. Such models help to understand this complex biological process and additionally can be used to reveal new targets for therapeutic antiviral agents.

Severe Acute Respiratory Syndrome (SARS) is a recently recognized epidemic disease characterized by significant morbidity and nearly 10% mortality. The etiologic agent is an enveloped RNA coronavirus. Coronaviruses are prevalent in nature and current findings suggest that the SARS eoronavirus entered the human population from animal reservoirs. Numerous studies have shown that the surface-exposed spike glycoproteins involved in coronavirus binding to host cells are critical factors in epidemiology and pathogenicity. Thus, it is likely that the spike proteins will have a major role in the disease of SARS-CoV infected patients. Therefore, the central objective of this project is to identify and characterize regions of the SARS spike proteins that confer the ability to infect both animals and humans. As part of this objective, we will identify host cell receptors used by SARS-CoV, using soluble spike proteins as detection reagents. As receptors are found, we will proceed by determining whether spike polymorphisms alter affinities for animal or human receptor homologs, as this may explain animal-to-human virus transmissions. We will focus on relevant human airway cells and will identify both cellular and viral determinants of spike-mediated syncytial formation, as syncytia are a recognized hallmark of coronavirus cytopathology. Coronaviruses can accommodate many adaptive mutations in their spike proteins, and we hypothesize that xenotropic and pathogenic SARS-CoVs are distinguished as a subset of these mutant forms. We will advance our understanding of SARS-CoV epidemiology and pathogenesis by identifying these variants and characterizing their receptor binding and syncytium inducing properties.

Coronaviruses cause respiratory and gastrointestinal diseases in birds and animals. These RNA viruses are genetically variable and can rapidly evolve to infect humans, sometimes causing severe acute respiratory disease. The coronaviruses are set apart in their assembly and secretion from cells by a strategy involving intracellular virus formation and perhaps novel trafficking routes to the outside environment. Further understanding of these late infection events will identify strategies for therapeutic intervention. We have discovered that a prototype mouse hepatitis coronavirus is inhibited at the assembly and / or secretion stages by very low nontoxic concentrations of a proteasome inhibitor. We hypothesize that the inhibitory mechanism involves ubiquitin depletion from cells, a known effect of proteasome inhibitors, because we discovered that the viral E proteins that are central to virus secretion are ubiquitinated on two lysine residues. Our aims are to determine whether pathogenic human coronaviruses are similarly hypersensitive to proteasome inhibitors and then address whether the ubiquitin conjugation of E proteins is central to coronavirus morphogenesis or expulsion out of cells. Our experiments will specifically evaluate whether the locus of inhibitor and ubiquitin action are at the virus secretion stages and will address the novel hypothesis that ubiquitin modifications direct the trafficking of virus-filled organelles to cell surfaces where the virus cargo is liberated. Our findings will reveal novel cell biological features of vesicle formation and organelle transport and will also inform us about the potential to thwart coronaviruses at late infection stages.

Human coronaviruses (H-CoVs) cause respiratory diseases ranging from self-limiting bronchiolitis to severe acute respiratory syndrome (SARS). This proposal aims to compare NL63-CoV, which causes mild disease, with SARS-CoV, focusing on distinctions between the viral spike (S) proteins that mediate the entry of these H-CoVs into host cells. The SARS and NL63 S proteins are divergent yet bind to the same host cell receptor, angiotensin converting enzyme 2 (ACE2). Subsequent to ACE2 interaction, these two S proteins may differentially direct virus entry and uncoating, in ways that may correlate with virus pathogenicity differences. Aim 1 addresses this question of distinct H-CoV entry pathways and extends preliminary data suggesting that integrins are used during NL63 S-mediated entry. Experiments will determine whether particular integrins are used by NL63 and other H-CoVs in tissue culture models of lung epithelia, and genetic approaches will be used to identify novel additional coreceptors participating in H-CoV entry. ACE2 operates as an ectopeptidase and as a signaling molecule to regulate the renin-angiotensin system and the inflammatory state of the injured lung. S proteins may disturb these central ACE2 functions. Aim 2 experiments will construct S fragments, recombinant SARS / NL63 S proteins, and HCoV-like particles bearing complete S proteins, for use as ACE2 ligands. S:ACE2 interactions leading to proinflammatory host cell responses will be identified, and the host responses to virus entry events operating subsequent to the ACE2 interaction will also be discerned. These studies relating S : ACE2 interactions with inflammation and lung pathology comprise a key collaboration with PPG component 3. The broader biological relevance of H-CoV entry cofactors and proinflammatory responses require the use of complete infectious viruses and host animal models. Robust in vitro and in vivo models to evaluate NL63-CoV are lacking. In Aim 3, we plan to select NL63-CoV variants for vigorous growth in culture, using cells overproducing ACE2 and integrins, with expectations for informative S mutants. Variants will be evaluated for growth in mice to establish models for the human disease. We will also construct recombinant NL63 / SARS chimeric viruses in collaboration with PPG component 4 to further correlate S proteins with virus growth, coreceptor usage and host cell responsiveness.

ABSTRACT Viruses need to be activated to infect cells. Activation in this context refers to a virus[unreadable] competence to uncoat and deposit genomes into cells, and is achieved by virus binding with cell receptors, virus acidification, reduction and / or virus proteolysis. Coronaviruses are enveloped RNA viruses causing respiratory diseases. Coronaviruses use a two-step activation strategy involving receptor binding followed by specific proteolysis. While a variety of cellular proteases can activate at this second step in the laboratory, it is not known whether a smaller subset of proteases operate normally in nature. Our central hypothesis in that the proteases relevant to coronavirus activation are those that are naturally (endogenously) linked to the primary virus receptors. We have evidence for a linkage between a transmembrane protease and a human coronavirus receptor and our proposal extends from these findings. Our first aim addresses the possibility that transmembrane proteases activate some coronavirus strains more readily than others, perhaps even before they bind receptors, and asks whether hyper-activation is characteristic of the most pathogenic virus strains. In this aim, we also propose to identify the precise time at which proteases cleave virus spikes. Our results will relate activation to virus virulence and will also give clearer pictures of activation processes. Our second aim is to evaluate connections between receptors and proteases in the more natural context of human lung-like cells and to expand analysis of receptor-protease complexes to the full range of known coronavirus receptors. In this aim, we also propose experiments to separate receptors from proteases and if possible, to find out whether such separation makes cells resistant to infection. Our results will potentially define virus-susceptible cells as those with intact receptor-protease complexes. Our third aim addresses the hypothesis that integrins operate as coreceptors for some coronaviruses. Our central hypothesis oversees this aim. The perspective is that integrins operate similarly to the known primary receptors, transporting bound viruses into regions with abundant activating proteases. This impact of this proposal will be to expand research in virus entry fields beyond studies of virusreceptor interactions and into studies of larger molecular complexes that include virus-activating proteases. This value and health relevance of this proposal will be to reveal new antiviral targets available during the virus entry process.

PROJECT SUMMARY The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic virus that can cause fatal disease in patients with underlying comorbidities. Further recognition of this respiratory syndrome and prevention strategies will require a small animal infection model as well as an additional understanding of the virus. This PPG describes a mouse model of MERS-CoV disease. In this model, the viruses causing disease are adapted variants, specialized for mouse lung infection. By contrast, non- adapted MERS-CoVs cause infection in the mouse but do not cause disease. The central hypothesis of this subproject (PPG2) is that mouse-adapted variants can efficiently enter host cells through pathways that are not available to the non-adapted viruses. To address this hypothesis, recombinant MERS-CoVs will be constructed and evaluated to determine whether mouse-adaptive mutations in the cell entry-mediating viral spike proteins correlate with efficient mouse lung infection. Surrogate MERS-CoV pseudo-viruses will be constructed and evaluated to address the focused hypothesis that mouse adapted variants mediate an ?early? plasma-membrane cell entry that is unavailable to non-adapted viruses. The project will dissect mechanisms by which spike proteins mediate early cell entry through plasma membranes versus late cell entry through endosomes. The basis for selection of early versus late cell entry will be determined by identifying host cell factors promoting or restricting either pathway. This project will also identify appropriate antiviral strategies that operate by preventing early and late virus-cell entry. The rationale for all of these aims is that additional understanding of MERS-CoV cell entry pathways will identify correlates of robust infection and disease, and will also provide insights on the best ways to prevent infection and disease with innovative virus entry inhibitors.

Abstract Recent studies by our group have revealed that the neuronal gene Arc, a master regulator of synaptic plasticity and information storage in the brain, acts as a repurposed retroviral Gag protein that forms capsids with the capacity to transmit genetic information between cells. These findings lead to a paradigm shift in the way we view both mechanisms of cognition and more generally how cells can signal to each other. This transformative R01 application will address these questions using a synergistic team of neuroscientists and virologists who will apply their expertise to Arc, intercellular gene transmission, and neuronal development. We will determine what genetic messages are transferred between neurons in Arc particles, how these particles enter ?target? neurons to deliver their RNA cargo to cell cytoplasm, and how delivery of this cargo influences the neuronal and synaptic processes that underlie memory and cognition. The methodologies to address these questions, as well as the potential impact of the answers, make this application ideally suited to the transformative R01 mechanism.