Title

Mechanisms of Mouse Hepatitis Entry into Cells

Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Susan T Hingley, PhD

Second Advisor

Brian J Balin, PhD

Third Advisor

Denah M Appelt, PhD

Fourth Advisor

Marcus G Bell, PhD

Abstract

Viruses can enter cells through several mechanisms, two common ones being
clathrin-mediated and caveolin-mediated endocytosis. The clathrin pathway delivers viral particles to endosomes, with subsequent acidification of the endosome and endosome/lysosome fusion often a prerequisite for release of the viral genome into the cytoplasm. The caveolin-mediated pathway delivers virus initially into vesicles called caveosomes, which have a neutral pH, before viral· uncoating occurs. Viral entry pathways can be examined by using various drugs to inhibit the different endocytosis pathways, as well as by siRNA technology to down-regulate expression of clathrin or caveolin proteins on the surface of host cells. To evaluate the possible importance of clathrin- and caveolin-mediated entry pathways for mouse hepatitis virus (MHV) entry into cells, an inhibitor of clathrinmediated endocytosis was used to inhibit infection with MHV. In addition, transfection with siRNA specific for clathrin or caveolin was used to monitor how down-regulation of these cellular proteins affects viral gene expression. The efficiency of siRNA transfection was determined by western blot analysis and real time reverse transcriptase polymerase chain reaction (RT2-PCR) using TaqMan gene expression assays. The abilities of recombinant virus expressing fusion proteins from two separate strains of MHV (MHVA59 and MHV-2) to enter both mouse fibroblast cells (L2 cells) and brain-derived endothelial cells (bEnd cells) was evaluated by RT2-PCR for viral specific sequences generated within the first round of viral replication. We confirmed that surface expression of caveolin and clathrin can be inhibited by siRNAs specific for these proteins. Down-regulation of these two proteins had a similar effect on viral gene expression for both strains of MHV. The inhibitor of clathrinmediated
endocytosis also demonstrated similar affects in the two cell lines for both strains of virus, but more extensive inhibition of viral replication than what was observed upon down-regulation of clathrin. Data from cells treated with inhibitors of clathrinmediated endocytosis, as well as from siRNA experiments, indicate that MHV can potentially utilize several mechanisms to enter cultured cells.

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