National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America.
PLoS One. 2012;7(4):e34761. doi: 10.1371/journal.pone.0034761. Epub 2012 Apr 4.
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-γ, IL-6, IL-12, iNOS and TNF-α) within 6 h. GSK-3β 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-3β in the context of encephalitic viral infections has been useful in a neuroprotective capacity. Small molecule GSK-3β inhibitors and GSK-3β siRNA experiments indicated that GSK-3β 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 IC(50) of ∼0.5 µM and a CC(50) of >100 µM. BIOder was a more potent inhibitor of GSK-3β than BIO, as demonstrated through in vitro kinase assays from uninfected and infected cells. Size exclusion chromatography experiments demonstrated that GSK-3β is found in three distinct complexes in VEEV infected cells, whereas GSK-3β 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-3β inhibitors for modulating VEEV infection.
甲病毒，包括委内瑞拉马脑炎病毒（VEEV），可导致马和人类发病，在很大比例的病例中表现出明显的脑炎。宿主免疫反应和组织特异性反应的特征可能导致致命结局以及脑炎的发展。先前已经表明，VEEV 感染小鼠在 6 小时内诱导促炎细胞因子基因（例如 IFN-γ、IL-6、IL-12、iNOS 和 TNF-α）的转录。GSK-3β 是一种宿主蛋白，已知其调节促炎基因表达，并且是阿尔茨海默病等神经退行性疾病的治疗靶点。因此，在脑炎病毒感染的情况下抑制 GSK-3β 在神经保护方面很有用。小分子 GSK-3β 抑制剂和 GSK-3β siRNA 实验表明 GSK-3β 对 VEEV 复制很重要。测试了 38 种第二代 BIO 衍生物，发现 BIOder 是最有效的抑制剂，IC50 约为 0.5 µM，CC50 大于 100 µM。通过未感染和感染细胞的体外激酶测定，BIOder 比 BIO 更能抑制 GSK-3β。排阻色谱实验表明，GSK-3β 在 VEEV 感染的细胞中存在于三个不同的复合物中，而在未感染的细胞中仅存在于一个复合物中。用 BIOder 处理的细胞表现出抗凋亡基因 survivin 的增加和促凋亡基因 BID 的减少，这表明调节促凋亡和抗凋亡基因有助于 BIOder 治疗的保护作用。最后，BIOder 部分保护了感染 VEEV 的小鼠免于死亡。我们的研究表明 GSK-3β 抑制剂在调节 VEEV 感染方面的效用。
