RESUMO
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans with high mortality. Not much is known about the interactions between viral and cellular factors that regulate JEV infection. By using a kinase/phosphatase-wide RNAi screening approach, we identified a cell cycle-regulating molecule, checkpoint kinase 2 (CHK2), that plays a role in regulating JEV replication. JEV infection induced G1 arrest and activated CHK2. Inactivation of CHK2 and its upstream ataxia-telangiectasia mutated kinase in JEV-infected cells by using inhibitors reduced virus replication. Likewise, JEV replication was significantly decreased by knockdown of CHK2 expression with shRNA-producing lentiviral transduction. We identified CHK2 as a cellular factor participating in JEV replication, for a new strategy in addressing JEV infection.
Assuntos
Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/isolamento & purificação , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/diagnóstico , Interferência de RNA , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Encefalite Japonesa/virologia , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno , Humanos , RNA Interferente Pequeno , Replicação ViralRESUMO
Virtually all eukaryotic cell functions and signaling pathways are regulated by protein phosphorylation. The Rad53 kinase plays crucial roles in the DNA damage response in Saccharomyces cerevisiae and is widely used as a surrogate marker for DNA damage checkpoint activation by diverse genotoxic agents. Most currently available assays for Rad53 activation are based on either electrophoretic mobility shifts or semiquantitative in situ autophosphorylation activity on protein blots. Here, we describe direct quantitative measures to assess Rad53 activity using immunoprecipitation kinase assays and quantitative mass spectrometric analysis of Rad53 activation loop autophosphorylation states. Both assays employ a highly specific Rad53 antibody, and thus enable the analysis of the untagged endogenous protein under physiological conditions. The principles of these assays are readily transferable to other protein kinases for which immunoprecipitation-grade antibodies are available, and thus potentially applicable to a wide range of eukaryotic signaling pathways beyond yeast.