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Degree

Doctor of Philosophy, University of Toledo, Biology (Cell-Molecular Biology), 2012.

Abstract

Interferons (IFN) are a family of cytokines characterized initially as antiviral proteins, but which have subsequently been implicated as protective molecules in malignant, inflammatory and immune disorders. The direct or indirect effects of IFN are mediated by a subset of genes known collectively as IFN stimulated genes (ISG), which are up regulated by IFNs via intracellular signaling cascade. The functions of some proteins encoded by ISGs are well described, but many other IFN induced proteins remained poorly characterized. Certain IFN induced proteins are characterized into different groups according to their amino acid sequence similarities, including the ISG12 group (6-16, ISG12a), 1-8 group (9-27/Leu13, 1-8U, and 1-8D), and ISG15. These IFN-induced genes are abundantly and widely expressed and induced mainly by type I IFNs. ISG12a encodes a predicted 11.5 kDa hydrophobic protein. Former studies from our laboratory have implicated ISG12a as a mitochondrial protein. Transient ISG12a expression augmented etoposide induced apoptosis by destabilizing the mitochondrial membrane. Knockdown of ISG12a prevented the sensitization to etoposide induced apoptosis and also decreased the ability of IFN-β pretreatment to sensitize cells to etoposide. In the present study, we assessed the function of ISG12a in cellular response to vesicular stomatitis virus (VSV) infection. We developed a conditional expression system for ISG12a using a modified Tet-on system. Interestingly, ISG12a expressing cells were protected from VSV when compared to control cells, and knocking down the ectopic protein with siRNA resulted in the loss of protection. Virus yield was reduced by ISG12a by approximately 105-fold and VSV mRNA expression was downregulated in ISG12 induced cells, suggesting a block in viral replication. To further analyze whether ISG12a blocks viral replication by upregulating IFN or other IFN regulated proteins, we performed RT PCR and affymetrix gene array studies on ISG12a expressing cells. Importantly, neither IFN nor IFN regulated genes were induced upon ISG12a expression, suggesting that ISG12a blocks viral replication in a different manner. To identify whether ISG12a inhibits VSV internalization, viral entry and binding assays were performed and viral genomic RNA was measured. VSV RNA levels were similar in control and ISG12a induced cells, suggesting that viral replication was blocked after internalization. To analyze whether ISG12a blocked endosomal release of viral particles into the cytoplasm, cells were treated with chloroquine (an inhibitor of endosome acidification) after VSV infection at different time intervals. VSV-G mRNA and protein levels were dramatically reduced in ISG12a induced cells in combination with chloroquine, suggesting that ISG12a probably blocks VSV replication at an early stage, either prior to or concomitant with endosomal release. Future studies will be focused on role of ISG12a in regulating mitochondrial function and whether this impacts viral replication.

Committee / Advisors

Douglas Leaman (Committee Chair)
Mark Wooten (Committee Member)
William Taylor (Committee Member)
Deborah Chadee (Committee Member)
Max Funk (Committee Member)

Document number: toledo1344217156
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=toledo1344217156

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