Ho Catherine M K, Donovan-Banfield I'ah Z, Tan Li, Zhang Tinghu, Gray Nathanael S, Strang Blair L
Institute of Infection & Immunity, St George's, University of London, Cranmer Terrace, London, SW17 0RE, United Kingdom.
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, United States of America.
PLoS One. 2016 Mar 1;11(3):e0150339. doi: 10.1371/journal.pone.0150339. eCollection 2016.
Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα.
蛋白激酶抑制剂可用作鉴定病毒复制所需蛋白质和信号通路的工具。通过病毒复制试验和蛋白质免疫印迹法，我们发现广泛使用的蛋白激酶抑制剂BAY61-3606可抑制人巨细胞病毒（HCMV）AD169株的复制以及AD169感染细胞中HCMV立即早期蛋白的积累，但对HCMV Merlin株的复制没有影响。通过体外激酶试验，我们发现BAY61-3606是细胞激酶IKKα的有效抑制剂。用靶向IKKα的小干扰RNA（siRNA）处理细胞后的感染结果表明，IKKα是AD169高效复制和立即早期蛋白产生所必需的。我们推测，作为经典核因子κB（NF-κB）信号通路的一部分，IKKα是AD169立即早期蛋白产生所必需的。然而，尽管BAY61-3606抑制了IKKα底物IκBα的磷酸化，但我们在AD169感染的细胞中未发现经典或非经典的NF-κB信号传导。相反，我们观察到在蛋白质免疫印迹试验中，用BAY61-3606或靶向IKKα的siRNA处理细胞会降低组蛋白H3丝氨酸10位点（H3S10p）的磷酸化水平。此外，我们发现用BAY61-3606处理细胞（而非用靶向IKKα的siRNA处理细胞）会抑制组蛋白H3乙酰化修饰（H3K9ac、H3K18ac和H3K27ac）和三甲基化修饰（H3K27me3和H3K36me3）的积累。因此，IKKα在HCMV复制中的需求具有毒株依赖性，并且在需要IKKα的HCMV毒株复制过程中，依赖IKKα的H3S10磷酸化与HCMV的高效复制和立即早期蛋白产生相关。此外，BAY61-3606对HCMV复制的抑制作用与不涉及IKKα的组蛋白H3乙酰化和三甲基化修饰有关。
