The mucosal surface is the site of entry of >90% of common infectious pathogens in humans. However, studies of virus entry and infection are largely limited to non-polarized cells and little is known regarding the mechanisms used by viral pathogens to infect polarized cell monolayers. Enteroviruses are ideal pathogens for the study of polarized cell-virus interactions as they have evolved highly efficient strategies to bypass both epithelial and endothelial barriers for infection. Our previous studies have established that coxsackievirus B (CVB) and poliovirus (PV) enter polarized cells by endocytic mechanisms that require activation of specific intracellular signaling molecules that drive actin cytoskeleton reorganization, junctional complex modulation, and eventual virus endocytosis. Our preliminary studies suggest that CVB endocytosis into polarized intestinal epithelia is distinct from that in polarized BBB endothelia and that the signaling molecules required to facilitate virus entry are disparate between these cell types. Based on these observations, we hypothesize that enterovirus entry into polarized epithelial and endothelial cells is mediated by cell-type-specific intracellular signals and occur through different endocytic mechanisms. In this proposal, we have designed experiments to more clearly define the cellular pathways hijacked by CVB to gain entry into polarized human brain microvascular endothelial cells (HBMEC), a model of the blood-brain barrier. Specifically, we will (1) define the endocytic pathway used by CVB to gain entry into HBMEC (Specific Aim 1), (2) characterize the role of intracellular kinases in facilitating CVB entry into HBMEC (Specific Aim 2), and (3) compare the intracellular signaling molecules required for enterovirus infection in a variety of cell types (Specific Aim 3). Given the diverse pathological complications associated with enterovirus infections, these studies will provide insights into how enteroviruses have evolved to infect polarized cells, and will serve as a model for how other viral pathogens circumvent the barriers presented by polarized cell monolayers. Furthermore, as there are currently no effective therapeutics to treat enterovirus infections, these studies may lead to the design of cell-type-specific anti-viral targets.
Enteroviruses (which include coxsackievirus, poliovirus, and echovirus) account for as many as 15 million symptomatic infections in the United States each year. There are currently no effective therapeutics to combat enteroviral infections. The studies in this proposal will identify cellular genes required for mediating enterovirus infection and may lead to the design of cell-type-specific antiviral targets.
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