Mechanisms underlying suppression of interferon α-induced anti-viral responses by the activated Ras/Raf/MEK pathway

Collier, Thaddeus William (2006) Mechanisms underlying suppression of interferon α-induced anti-viral responses by the activated Ras/Raf/MEK pathway. Masters thesis, Memorial University of Newfoundland.

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Abstract

Interferon α (IFN) is important for anti-viral defense. IFN binds to cell surface receptors and stimulates the Janus kinase-signal transducer and activator of transcription (Jak/STAT) pathway which activates transcription of anti -viral effector genes 2' ,5 '-oligoadenylate synthetase (OAS), myxovirus resistance gene 1 (Mx1), and double stranded RNA-dependent protein kinase (PKR). Upon virus infection, viral double stranded RNA (dsRNA) activates the OAS/latent ribonuclease (RNase L) pathway which degrades viral RNA whereas the PKR/eiF2α pathway blocks translation of viral RNA. Mx1 has been shown to be involved in blockage of viral nucleocapsid transport or viral RNA synthesis. Previously, we demonstrated that activation of the Ras/Raf/MEK pathway inhibits IFN-induced anti-viral responses. The objective of this study was to determine how activated Ras/Raf/MEK pathway suppresses IFN-induced responses. First, we stimulated vector control NIH 3T3 cells (BABE) and Ras transformed NIH 3T3 cells (RasV12) with IFN and then examined mRNA expression levels of OAS, Mx1, and PKR mRNA at 6 and 12 hours after the stimulation. We found that OAS, Mx1, PKR mRNA expression were higher in BABE compared to RasV12. We were able to rescue Mx1 and OAS gene expression induced by IFN in RasV12 by treatment of U0126, a specific inhibitor of MEK. Similarly, western blot analysis revealed that OAS protein levels were restored in RasV12 stimulated with IFN using U0126 and RNA interference (RNAi) to Ras. To further determine how activated Ras/Raf/MEK pathway suppresses IFN-induced gene transcription, we investigated activation status of the Jak/STAT pathway in IFN stimulated BABE and RasV12 by Western blot analysis using antibodies against phosphorylated STAT1, total STAT1, phosphorylated STAT2 and total STAT2. As a result, we observed higher phosphorylation and total levels of STAT1 and STAT2 in BABE compared to RasV12. When treated with U0126, phosphorylation of both STAT proteins was restored in RasV12. These results suggest that activated Ras/Raf/MEK pathway inhibits activation of the JAK/STAT pathway leading to suppression of IFN-induced gene transcription.

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