Viral Pathogenesis and Regulation of Innate Immunity
Viruses successfully negotiate a gauntlet of cellular restrictions that serve to limit their entry, replication, assembly and dispersal. In the process viruses alter cellular responses that contribute to pathogenesis. Viral entry is a key determinant of host, tissue and cellular tropism and often delimits the intrinsic characteristics of viral disease. Upon entry successful viral pathogens regulate innate immune defenses that normally limit viral replication and spread. In addition, virally altered cell signaling pathways, transcriptional responses and receptor functions induce changes in cellular functions that contribute to pathogenesis.
Our lab is focused on defining mechanisms of pathogenesis resulting from viral protein interactions with receptors and signaling pathways that alter normal endothelial cell responses. Endothelial cells form a dynamic fluid barrier within capillaries that balances the influx and efflux of solutes with tissues, regulates immune cell extravasation and directs vessel repair while maintaining vascular integrity through redundant failsafe mechanisms. Hantaviruses and dengue viruses nonlytically cause hemorrhagic and edematous diseases by altering fluid barrier functions of the endothelium.
Hantavirus Pathogenic Mechanisms and Regulation of Innate Immunity:
||Fig.1 Calcein labeled platelet binding to the surface of mock, HTNV, ANDV or TULV infected ECs, 3 days p.i.
Pathogenic hantaviruses primarily infect endothelial cells and cause two vascular leak syndromes, hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Human β3 integrins play prominent roles in regulating vascular permeability through platelets and endothelial cells. On endothelial cells β3 integrins temper the permeabilizing effects of vascular endothelial growth factor (VEGF) by binding to VEGF receptor 2 (VEGFR2).
We determined that HPS and HFRS causing hantaviruses bind inactive conformations of β3 integrins and enhance endothelial cell responses to VEGF. Elevated VEGF in pulmonary edema fluids of HPS patients contributes to hypoxia and the likely induction of an autoamplifying VEGF-HIF1a loop that contributes to the accumulation of up to 1 liter per hour of pulmonary edema fluid. We are currently investigating the role of hantavirus proteins, signaling pathways and altered VEGFR2 and β3 interactions as mechanisms of hantavirus pathogenesis and as potential therapeutic targets to restore endothelial cell barrier functions. We are also addressing the mechanism by which hantaviruses alter endothelial cells microRNAs which regulate fluid barrier functions and defining the role of viral N and pol proteins in altered miRNA and signaling responses.
Lymphatic vessels clear fluid from tissues and are lined by uniquely regulated lymphatic endothelial cells (LECs). Our findings indicate that LECs are both infected by hantaviruses and uniquely altered by ANDV infection which results in the formation of giant cells. Our findings tie rapamycin sensitivity of LEC giant cells to altered mTOR-S6K activation and hypoxia driven responses. We are studying the mechanism by which mTOR is activated by hantavirus proteins in primary LECs and defining potential therapeutic targets that may resolve fluid clearance deficits in HPS patients.
Pathogenic viruses inhibit the early induction of IFN in order to replicate within cells. We are currently analyzing hantavirus proteins that regulate pathway specific responses directed by RIG-I-MAVS-TRAF3-TBK1 signaling pathway that block IRF-3 and NF-kB activation. A virulence determinant within the hantavirus Gn-tail protein is being defined and has the potential to attenuate pathogenic hantaviruses using reverse genetic approaches that are being developed.
Dengue Virus: Role of Endothelial Cells in Dengue Infections
Dengue viruses (DVs) cause 2 vascular leak syndromes: dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). However, DVs primarily infect immune cells and instead of a direct effect on vascular permeability, infection by a second DV serotype gives rise to an immune enhanced disease process resulting in hemorrhage or edema in a small percentage of patients. Ultimately vascular permeability is the result of changes in the vascular endothelium. We have demonstrated that primary human ECs are efficiently and productively infected. DV infected endothelial cells rapidly produce virus and appear to regulate early but not late IFN responses. Interestingly our findings demonstrate that DV infected ECs also secrete cytokines and chemokines that have the potential to contribute to immune enhanced pathogenesis. In fact the presence of DV antigen in endothelial cells provides a potential target for enhanced immune responses during secondary DV infections.
Our lab is interested in determining the contribution of endothelial cells to immune enhanced DV responses and viremia observed in dengue patients. Because dengue virus replicates quickly and efficiently to high titers in endothelial cells, we are also interested in the mechanisms by which DV non-structural proteins inhibit early interferon signaling pathways that allows viral replication. These studies are aimed at defining viral and cellular targets for therapeutically regulating DV responses that contribute to the severity of disease in dengue patients.
Additional Studies: Studies of rotavirus regulation of cellular responses, molecular mechanisms of pathogenesis, reverse genetics and rotavirus protein function are continued interests of the lab as well as the development of reverse genetics systems for hantaviruses.