蛋白激酶A通过磷酸化埃博拉病毒VP35调控病毒复制

Protein Kinase A Mediated Phosphorylation of Ebola Virus VP35 Regulates the Viral Replication

埃博拉病毒病(Ebola virus disease,EVD)是由埃博拉病毒(Ebola virus,EBOV)感染所引起的急性出血性伴多脏器损害的烈性传染病。VP35蛋白是EBOV RNA合成必需的聚合酶辅助因子,并可通过阻断干扰素途径抑制机体先天性免疫,其磷酸化可以促进病毒复制。我们前期研究发现EBOV VP35与A激酶相互作用蛋白1(A⁃kinase interacting protein 1,AKIP1)相互作用并激活蛋白激酶A(Protein kinase A,PKA),但PKA是否可磷酸化VP35,从而调控EBOV复制尚不清楚。本研究发现,PKA激活剂Forskolin(FSK)及8⁃Bromo⁃cAMP促进VP35丝氨酸磷酸化,而PKA抑制剂H89及蛋白激酶抑制剂肽(Protein kinase inhibitor peptide,PKI)抑制VP35磷酸化。通过质谱分析鉴定出VP35存在多个磷酸化位点,VP35 S187A突变后,VP35磷酸化显著减弱,且PKA激活剂FSK和8⁃Bromo⁃cAMP不能促进VP35 S187A的磷酸化,提示S187是PKA介导VP35磷酸化的主要位点。接着,利用可模拟病毒生命周期的EBOV trVLP系统研究了VP35磷酸化对病毒复制的影响,发现携带VP35 S187A突变的EBOV trVLP在细胞中的复制降低17倍,而H89和PKI处理并不能进一步抑制EBOV trVLP的增殖。这些结果表明,PKA通过介导VP35 S187位的磷酸化,促进EBOV trVLP的复制。此外,VP35 S187不影响VP35拮抗干扰素β产生的功能。本研究发现PKA通过磷酸化VP35 S187调控EBOV的复制,提示PKA抑制剂可以用于拮抗EBOV的增殖,进而为EVD的治疗提供新思路。

Ebola virus disease(EVD)is an acute hemorrhagic disease with multi⁃organ damage caused by Ebola virus(EBOV)infection.VP35 protein is an essential polymerase cofactor for EBOV RNA synthesis,which can inhibit innate immunity by blocking the type I interferon pathway,and its phosphorylation can promote viral replication.Our previous work demonstrated that the EBOV VP35 interacts with AKIP1 and activates protein kinase A(PKA),but whether VP35 can be phosphorylated by PKA has not been investigated.In this study,PKA inhibitors(H89,PKI)and PKA activators(FSK and 8⁃Bromo⁃cAMP)were used to treat VP35⁃expressing cells,and the results showed that VP35 phosphorylation could be potentiated by PKA activators FSK and 8⁃Bromo⁃cAMP,or suppressed by PKA inhibitors H89 and PKI.Next,phosphorylation of VP35 was observed to be substantially suppressed by the mutation of S187 to A.PKA activator FSK and 8⁃Bromo⁃cAMP showed little if any effect on phosphorylation of VP35 S187A,suggesting that S187 is the main site of PKA⁃mediated VP35 phosphorylation.Furthermore,viral replication was evaluated by trVLP,a system simulating full life cycle of EBOV.EBOV trVLP in which VP35 was replaced by S187 to A showed 17 folds suppression of viral replication,and PKA inhibitors H89 and PKI showed little if any effect on the replication of trVLP bearing VP35 S187A.These results collectively suggested that PKA mediated phosphorylation of viral VP35 S187 potentiates the replication of EBOV trVLP,indicating that PKA promoted EBOV trVLP replication by mediating phosphorylation at the VP35 S187 site.Moreover,VP35 S187A mutation showed little if any effect on the antagonism function of VP35 on interferon beta(IFN⁃β)production.In summary,this study demonstrated PKA mediated phosphorylation of viral VP35 at S187 potentiates EBOV replication.PKA inhibitors treatment or blocking VP35 phosphorylation may be potential to antagonize the proliferation of EBOV,thus providing a new approach for the development of therapeutics for EVD.