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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI081759-04
Application #
8295004
Study Section
Virology - A Study Section (VIRA)
Program Officer
Park, Eun-Chung
Project Start
2009-07-01
Project End
2013-09-05
Budget Start
2012-07-01
Budget End
2013-09-05
Support Year
4
Fiscal Year
2012
Total Cost
$356,070
Indirect Cost
$111,045
Name
University of Pittsburgh
Department
Physiology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Bayer, Avraham; Delorme-Axford, Elizabeth; Sleigher, Christie et al. (2015) Human trophoblasts confer resistance to viruses implicated in perinatal infection. Am J Obstet Gynecol 212:71.e1-8
Harris, Katharine G; Coyne, Carolyn B (2014) Death waits for no man--does it wait for a virus? How enteroviruses induce and control cell death. Cytokine Growth Factor Rev 25:587-96
Delorme-Axford, Elizabeth; Morosky, Stefanie; Bomberger, Jennifer et al. (2014) BPIFB3 regulates autophagy and coxsackievirus B replication through a noncanonical pathway independent of the core initiation machinery. MBio 5:e02147
Mouillet, Jean-Francois; Ouyang, Yingshi; Bayer, Avraham et al. (2014) The role of trophoblastic microRNAs in placental viral infection. Int J Dev Biol 58:281-9
Ouyang, Y; Mouillet, J-F; Coyne, C B et al. (2014) Review: placenta-specific microRNAs in exosomes - good things come in nano-packages. Placenta 35 Suppl:S69-73
Jacobs, Jana L; Zhu, Jianzhong; Sarkar, Saumendra N et al. (2014) Regulation of mitochondrial antiviral signaling (MAVS) expression and signaling by the mitochondria-associated endoplasmic reticulum membrane (MAM) protein Gp78. J Biol Chem 289:1604-16
Harris, Katharine G; Coyne, Carolyn B (2013) Enter at your own risk: how enteroviruses navigate the dangerous world of pattern recognition receptor signaling. Cytokine 63:230-6
Delorme-Axford, Elizabeth; Sadovsky, Yoel; Coyne, Carolyn B (2013) Lipid raft- and SRC family kinase-dependent entry of coxsackievirus B into human placental trophoblasts. J Virol 87:8569-81
Delorme-Axford, Elizabeth; Donker, Rogier B; Mouillet, Jean-Francois et al. (2013) Human placental trophoblasts confer viral resistance to recipient cells. Proc Natl Acad Sci U S A 110:12048-53
Bozym, Rebecca A; Delorme-Axford, Elizabeth; Harris, Katharine et al. (2012) Focal adhesion kinase is a component of antiviral RIG-I-like receptor signaling. Cell Host Microbe 11:153-66

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