Adenovirus infection is dependent on regulation and accessibility of the receptor car .

Coxsackie and Adenovirus Receptor (CAR) displays dual biological functions since it acts both as a viral receptor for the two unrelated viruses, coxsackie and adenovirus (Ad), and as a cell-adhesion molecule in mammalian cells. The number and accessibility of CAR receptors expressed at the cell surface is a major determinant for successful infection. The aim of this thesis is to evaluate how CAR and Ad infection vary depending on the physiological milieu surrounding the cell. To address the aim of the thesis, CAR-mediated Ad infection was studied under three different conditions, namely in the presence of anti-Ad neutralizing antibodies that constitute a humoral immune response to the viral capsid, in the presence of cytokines that constitute secreted pro-inflammatory mediators, and in the presence of altered intracellular signaling pathways that constitute hallmarks of cancer progression. To examine the impact of neutralizing antibodies on CAR-mediated Ad infection, quantitative methods were developed to measure cell-associated virus and successful viral infection (gene expression). In the presence of neutralizing antibodies, Ad was hindered from interaction with CAR and infection was prevented. The impact of neutralizing antibodies was further characterized by determining the extent to which the decrease in infection resulted from hindered receptor binding versus actual viral inactivation. To examine this question, target cells were modified by introducing the expression of an Fcγ receptor that was capable of binding and internalizing Ad-antibody complexes in a CAR-independent manner. These experiments showed that infectious virus was present in Ad-antibody complexes and that hindrance of binding to CAR likely constituted a major factor in neutralizing Ad. To examine the impact of inflammation on Ad infection, CAR expression and Ad infection of human endothelial cells were studied in the presence and absence of the proinflammatory cytokines, tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). The data showed that these cytokines suppressed CAR protein and mRNA levels in endothelial cells and inhibited Ad infection in a time and dose-dependent manner, demon-strating that cytokine-mediated changes in cell physiology had the potential to affect CAR-dependent Ad infection by changing the availability of CAR. Finally, to examine the impact of cancer-related intracellular signaling pathways on CAR-mediated Ad infection, several in vitro models were established to recreate progression of tumor cells from low to high-grade malignancy, a process known as epithelial to mesenchymal transition (EMT). CAR was suppressed both at the transcriptional and translational level in these models, and a novel transcriptional repressor complex involving Snail and Smads was identified. This complex mediated effective suppression of CAR as well as another cell adhesion protein E-cadherin reflecting the fact that CAR expression and Ad infection can be modulated as part of larger, long term changes in cell physiology. In summary, these thesis studies shed new light on mechanisms involved in adenovirus interaction with host cells and on regulation and accessibility of CAR during normal and pathological conditions. As such this thesis has, in part, contributed to a better understanding of the intimate interplay between virology and cell biology. f Abbreviations aa amino acid Ad adenovirus ADE antibody-dependent enhancement ALK5 activin receptor-like kinase 5 AP-1 activator protein 1 APC antigen presenting cell β-gal β-galactosidase BT-IgSF brain and testis immunoglobulin superfamily CAR coxsackie and adenovirus receptor CBP CREB-binding protein ChIP chromatin immunoprecipitation cIAPs cellular inhibitors of apoptosis CLMP CAR like membrane protein CMV cytomegalovirus co-Smad cofactor Smad CPE cytopatic effect CR3 complement receptor 3 CTL cytotoxic T-lymphocyte CTX Cortical Thymocyte in Xenopus DC dendritic cells ECM extracellular matrix EGF epidermal growth factor EMT epithelial-mesenchymal transition ERK extracellular signal-regulated kinase ESAM endothelial cell selective adhesion molecule E-selectin endothelial-selectin FADD fas-associated death domain protein FAK focal adhesion kinase FasL Fas Ligand FcR Fc receptor FGF fibroblast growth factor FGFR FGF receptor GAS IFNγ activating sites GSK3β glycogen synthase β H-cadherin heart-cadherin HGF hepatocyte growth factor HIV human immunodeficiency virus H-Ras Harvey-Ras HUVEC human umbilical vein cells ICAM intercellular adhesion molecule IFNGR1 interferon gamma receptor 1 Ig immunogloubulin IgSF immunoglobulin superfamily Iκκ inhibitor of kappa β kinase IL-2 Interleukin-2 ILK integrin-linked kinase INFγ interferon gamma I-Smad inhibitory Smad ITAM Immunoreceptor Tyrosine-based Activation Motif ITIM Immunoreceptor Tyrosine-based Inhibitory Motif JAK1 janus activated kinase 1 JAM junctional adhesion molecule JAM-L JAM-like K-cadherin kidney-cadherin kDa kilodaton K-Ras Kirsten-Ras LNX ligand-of-numb-protein X LOXL2 lysyl oxidase like protein 2 L-selectin leukocyte selectin MAG1-1β membrane–associated guanlyate kinase 1β MAPK Mitogen-Activated Protein Kinase MEK1 mitogen-activated protein kinase and ERK kinase 1 MET mesenchymal epithelial transition MHC major histocompatibitity complex MIP-1α macrophage inflammatory protein-1α MLCK myosin light chain kinase MMP matrix metalloproteinase MTOC microtubule organizing center MTs microtubules MUPP-1 multi-PDZ domain protein-1 N-cadherin neural-cadherin NFκβ nuclear factor kappa beta NK cells natural killer cells NLS nuclear localization signal N-Ras Neuroblastoma-Ras P-cadherin placental cadherin PDK1 phosphoinositide-dependent kinase 1 PDZ PSD95/DLG/ZO-1 PECAM platelet/endothelial cell adhesion molecule 1 PI3K phosphatidyl inositol phosphatase kinase PICK1 protein interacting with protein C kinase PIP3 phosphatidylinositol 3, 4, 5 triphosphate PKC protein kinase C P-selectin platlet-selectin PTEN phosphatase and tensin homolog R-cadherin retinal-cadherin RGD Arg-Gly-Asp RIP1 receptor interacting protein 1 R-Smad receptor Smad SARS severe acute respiratory syndrome SBEs Smad binding elements SEAP secreted alkaline phosphatase SODD silencer of death domains STAT1 signal transducers and activators of transcription TER transepithelial electrical resistance TGFβ tumor growth factor beta TNFα tumor necrosis factor alpha TNFR1 tumor necrosis factor receptor 1 TRADD TNFR1 associated death domain protein TRAIL TNF related apoptosis inducing ligand TRAF2 TNF-receptor-associated factor 2 VCAM vascular cell adhesion molecule 1 VE-cadherin vascular endothelial cadherin WT wild-type ZO zona occludens ZONAB ZO-1-associated nucleic acid-binding protein