Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Stephanie DeWitte-Orr

Advisor Role

Supervisor

Abstract

When infecting cells, most viruses produce dsRNA molecules at some point in their replicative cycle. When an infection results in cell lysis, these molecules are released into the extracellular space. Class A scavenger receptors (SR-As) on the surface of animal cells bind extracellular dsRNA and bring it into the cell where dsRNA triggers an innate immune response, enabling the cell to protect itself from an impending virus infection. Very few cells are unable to bind and respond to extracellular dsRNA, thus cell-based assays for studying extracellular dsRNA sensing via SR-As have been limited. CHSE-214 cells are a promising model for SR-A study as preliminary reports suggest they are unable to initiate an antiviral response when treated with extracellular dsRNA, but are able to respond to intracellular dsRNA (via transfection); possibly due to a lack of functional SR-As. Using qRT-PCR to measure the expression of innate immune gene transcripts and a cytopathic effect assay to investigate whether immune gene expression leads to an antiviral state, the present study found that CHSE-214 cells express immune genes and establish an antiviral state only when dsRNA is transfected directly into the cell, not in response to extracellular dsRNA. Immunocytochemistry showed that CHSE-214 cells cannot bind or internalize dsRNA, furthering the hypothesis that they may lack functional SR-As. To further investigate the feasibility of CHSE-214 functioning as a cell model for SR-A binding and signaling, novel rainbow trout SR-A sequences were cloned into expression vectors and sequenced. Bioinformatics analysis shows that these sequences have similar protein architecture to those in other species and phylogenetic analysis shows a strong relationship to other fish MARCO and SCARA5 sequences. Preliminary data have shown that overexpression of exogenous SR-As in CHSE-214 enables it to bind extracellular dsRNA. Developing a cell line for use as a model for the study of class A scavenger receptors will facilitate the understanding of these receptors’ functions in innate immunity.

Convocation Year

2017

Convocation Season

Spring

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Cell Biology Commons

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