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EFFECTS OF INTERFERON ALPHA ON
HEPATITIS C VIRAL TRANSLATION
by
Ronald Jubin
Department of Microbiology & Molecular Genetics
M.S., 1995, Seton Hall University
Thesis Advisor: Harvey Ozer, M.D.
Senior Associate Dean for Research and
Professor
Department of Microbiology & Molecular Genetics
ICPH Auditorium
225 Warren Street
Monday, March 29, 2004
12:00 Noon
Abstract
Interferons are critical components of the host innate immune response. Numerous infectious agents including viruses invoke cellular production and secretion of interferons. Binding of a respective IFN with its cognate receptor initiates a complex signal cascade that renders the cell refractory for viral replication. Several cellular proteins including protein kinase R (PKR) are essential factors critical to a robust antiviral response. PKR is a dsRNA-activated kinase. Interaction with dsRNA promotes auto-phosphorylation and subsequent trans-phosphorylation of eIF2a reducing overall protein synthesis rates. Hepatitis C infection affects over 170 million people worldwide. Current therapy comprised of pegylated interferon alpha (IFNa) and ribavirin is only 50% effective in treating genotype 1 infected patients. The mechanism of Hepatitis C virus (HCV) sensitivity to IFNa is unclear but likely involves both viral and host factors. Specifically, the exact role of PKR in IFNa-mediated control HCV has been controversial, with studies indicating both PKR-dependent and independent mechanisms. HCV viral translation is under control of an internal ribosome entry site (IRES) that possesses a unique strategy for translation initiation. In this study, the rabbit reticulocyte lysate system was used to discern the role of PKR in regulating HCV translation in the absence of de novo gene expression. This analysis was accomplished by pre-treatment of lysates with the PKR agonist poly I:C or antagonists 2-aminopurine or Adenovirus VAI RNA. Subsequently, studies were performed in Huh7 cell lines harboring integrated bi-cistronic plasmid DNAs containing HCV IRES. Finally, studies were performed in Huh7 cells containing stably selected HCV replicons. Rabbit reticulocyte studies revealed that the HCV IRES was sensitive to PKR activation. Surprisingly, PKR antagonists alone enhanced HCV IRES translation, suggesting that the HCV IRES auto-regulates its own translation efficiency in a PKR-dependent manner. Cell-based studies showed that co-addition of the PKR antagonist 2-AP could delay the inhibitory effects of IFNa on HCV replicon RNA levels, suggesting that PKR plays an important role in the early control of HCV post-IFNa treatment. PKR-regulated viral protein expression may represent an important event for viral pathogenesis and establishment of chronic infection.
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