The Molecular Basis of the Interaction Between the Coxsackievirus and Adenovirus Receptor (CAR) and MAGI-1

Author

Abimbola Olayinka Kolawole, Wright State University

Publication Date

2011

Document Type

Dissertation

Committee Members

Nancy Bigley (Committee Member), Katherine Excoffon (Advisor), Michael Leffak (Committee Chair), Mill Miller (Committee Member), Robert Putnam (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

A major factor in virus entry into cells is localization and abundance of the primary receptor. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for group B coxsackievirus and many serotypes of adenovirus. In most epithelia, a seven exon isoform of CAR (CAR^Ex7) is exclusively localized at the basolateral surface where it behaves as a homophilic adhesion protein and is inaccessible for viral infection. However, in well-differentiated human airway epithelia, we recently discovered an alternatively spliced, low abundance isoform of CAR (CAR^Ex8) that is apically localized where it may initiate apical viral infection. The two isoforms differ only in the last 26 (CAR^Ex7) or 13 (CAR^Ex8) amino acids of the cytoplasmic domain, which suggests that some intracellular interactions may differ. One such differential interaction involves MAGI-1, an essential PDZ-domain containing protein known to be involved in cell polarization and cancer. In non-polarized COS7 cells, the CAR^Ex8 protein level is regulated by MAGI-1b. CAR-MAGI-1 interactions were investigated by MAGI-1 siRNA knockdown, in vitro translation, immunocytochemistry, co-immunoprecipitation-Western blot analysis, fluorescence resonance energy transfer, direct binding assays, and adenovirus infection. Data showed that both CAR isoforms were expressed in several cell lines with CAR^Ex7 RNA consistently more highly expressed than CAR^Ex8 and MAGI-1 siRNA knockdown improved adenovirus infection in polarized cells. Both isoforms strongly interacted with PDZ3. CAR^Ex8 also interacted with PDZ1. Whereas co-expression of PDZ1 with CAR^Ex8 did not affect adenovirus infection, it could compete with full length MAGI-1 to protect CAR^Ex8 from loss. Co-expression of CAR^Ex8 with PDZ3 significantly reduced CAR^Ex8 cell surface expression and adenovirus infection but did not reduce total CAR^Ex8 protein levels suggesting that this may be the MAGI-1 PDZ domain responsible for holding CAR^Ex8 within the cell but it is likely that other MAGI-1 interacting proteins/domains are required for loss of CAR^Ex8. This dissertation provides the molecular basis for MAGI-1-mediated regulation of CAR^Ex8 which will allow further investigation into the mechanisms of CAR^Ex8 cell surface expression and hence viral infection of polarized epithelia.

Page Count

167

Department or Program

Biomedical Sciences

Year Degree Awarded

2011

Copyright

Copyright 2011, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.