Title

Investigation of Mouse Hepatitis Virus Entry Using Small Interfering RNA Technology

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. Caveolin-mediated entry delivers virus to vesicles called caveosomes, which have a neutral pH. Mouse hepatitis virus strain A59 (MHV-A59) is a fusogenic coronavirus that can induce virus-cell fusion in mouse fibroblast cells (L2 cells) at neutral pH and can potentially enter L2 cells through caveolin-mediated endocytosis. MHV -2 is a nonfusogenic coronavirus that has been shown to require cleavage by lysosomal cathepsins, which require low pH for activity, in order to mediate entry; MHV-2, therefore, is dependent on clathrin-mediated endocytosis for entry. Studies of viral entry typically use various drugs to inhibit the different endocytosis pathways. However, it is possible that these drugs have multiple affects on host cells that influence several steps of the viral replication cycle, not just viral entry. To better evaluate the importance of both clathrin- and caveolin-mediated entry mechanisms, we used siRNAs specific for clathrin and caveolin sequences to down-regulate expression of these proteins on L2 cells. The efficiency of siRNA transfection was determined by western blot analysis, immunofluorescence labeling and real time reverse transcriptase polymerase chain reaction (RT2-PCR) using TaqMan gene expression assays. The abilities of MHV-A59 and MHV -2 to enter L2 cells expressing decreased levels of clathrin or caveolin was evaluated by RT2-PCR for viral specific sequences. Through siRNA down-regulation of caveolin, our RT2-PCR data suggest that both MHV-A59 and MHV-2 use caveolin for entry into cells. RT2-PCR analysis with caveolin siRNA revealed that RA59sAs9 entry was decreased by an average of 44% and RA59s2 entry by an average of 56%. Studies of other cell lines, RT2-PCR analysis of clathrin mRNA and viral entry via clathrin mediated endocytosis are still in progress. The use of siRNA technology should provide for a more direct evaluation of the relative importance of clathrin- or caveolin-mediated endocytosis in viral entry.

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