Modulation of GSK-3β Activity in Venezuelan Equine Encephalitis Virus Infection

Alphaviruses, including Venezuelan Equine Encephalitis Virus (VEEV), cause disease in both equine and humans that exhibit overt encephalitis in a significant percentage of cases. Features of the host immune response and tissue-specific responses may contribute to fatal outcomes as well as the development of encephalitis. It has previously been shown that VEEV infection of mice induces transcription of pro-inflammatory cytokines genes (e. g., IFN-gamma, IL-6, IL-12, iNOS and TNF-alpha) within 6 h. GSK-3 beta is a host protein that is known to modulate pro-inflammatory gene expression and has been a therapeutic target in neurodegenerative disorders such as Alzheimer's. Hence inhibition of GSK-3 beta in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3 beta inhibitors and GSK-3 beta siRNA experiments indicated that GSK-3 beta was important for VEEV replication. Thirty-eight second generation BIO derivatives were tested and BIOder was found to be the most potent inhibitor, with an IC50 of similar to 0.5 mu M and a CC50 of >100 mu M. BIOder was a more potent inhibitor of GSK-3 beta than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3 beta is found in three distinct complexes in VEEV infected cells, whereas GSK-3 beta is only present in one complex in uninfected cells. Cells treated with BIOder demonstrated an increase in the anti-apoptotic gene, survivin, and a decrease in the pro-apoptotic gene, BID, suggesting that modulation of pro-and anti-apoptotic genes contributes to the protective effect of BIOder treatment. Finally, BIOder partially protected mice from VEEV induced mortality. Our studies demonstrate the utility of GSK-3 beta inhibitors for modulating VEEV infection.