Abstract
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.
Highlights
Cellular protein kinases are required for many aspects of viral replication and pathogenesis
We employed viral yield reduction and viral plaque reduction assays to assess the ability of BAY61-3606 to inhibit replication of human cytomegalovirus (HCMV) strain AD169 in human foreskin fibroblast (HFF) cells
To exclude the possibility that the observed reduction in HCMV replication is due to BAY61-3606 toxicity in HFF cells, we exposed HFF cells to BAY61-3606 at a range of concentrations and used an MTT dye-uptake assay to assess cell viability
Summary
Cellular protein kinases are required for many aspects of viral replication and pathogenesis. During human cytomegalovirus (HCMV) replication cellular proteins kinases have prominent roles in genome replication as intracellular signaling pathways that contain protein kinases have been implicated in activation of viral and cellular transcription during productive replication [1]. Productive HCMV transcription from the viral genome first requires activation of the HCMV immediate early promoter (MIEP), which controls production of the immediate-early viral proteins IE1 and IE2 [1]. These proteins stimulate a transcriptional cascade of immediateearly to early to late RNA transcripts required for production of infectious virus [1]. The MIEP is a complex promoter that can be recognized by transcription factors controlled by intracellular signaling pathways containing cellular protein kinases [1]. Most controversial, signaling pathway reported to be involved viral transcription is the canonical NF-κB signaling pathway
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
