疱疹病毒丝氨酸/苏氨酸蛋白激酶参与激活潜伏感染Epstein-Barr病毒（EBV）的分子机制研究

Lantency is the characteristic feature of herpesviruses. Being an important human herpesvirus, the reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation EBV is not yet fully understood. It is clinically evident that many herpesviruses (e.g. HSV) co-infect with EBV. We have reported that HSV induced the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post-HSV-1 infection in virus-entry-dependent and replication-independent manners. All herpesviruses encode two types of Ser/Thr kinases. The prototypical examples are HSV UL13 and Us3, both are viral tegument proteins. We have previously shown that the "HSV-PKA-CREB-BZLF1" pathway is important for HSV infection induced EBV reactivation. HSV US3 mimics PKA. It shares phosphorylation target sequences with PKA, and has been reported to activate PKA by phosphorylation. Indeed, our results indicated that US3 similarly behaves with PKA in transient transfection assays. The overexpression of US3 greatly enhanced the CREB phosphorylation level, and this enhancement depended on kinase activity. UL13 is the kinase for Us3. Based on these results, we hypothesize that the UL13 and US3 kinase of HSV can reactivate EBV Zp through the phosphorylation of CREB. Further, we propose that this mechanism is conserved within the herpesvirus family. In this study, on one hand, we will use HSV-BAC recombination sysytem to generate HSV-1 US3 and UL13 single/double null-mutant viruses, on the other hand, we will express recombinant US3 and UL13 and its homologues from the other herpesvirus in EBV latently infected cells. This study will be valuable to answer wether this mechanism could be serving as a target for therapeutic intervention.

潜伏感染是EBV等疱疹病毒的特征。EBV可引起鼻咽癌和淋巴瘤等重大疾病，潜伏感染的激活与其致病机制密切相关。临床研究显示HSV等疱疹病毒可与EBV共感染。我们发现，HSV的超感染可激活潜伏感染的EBV。因此，疱疹病毒的共感染可能是激活潜伏EBV的诱因。我们前期研究发现"HSV-PKA-CREB-BZLF1"是HSV感染激活潜伏EBV的重要通路（已发表）。HSV可在感染6小时内高效激活EBV，提示该激活不依赖于病毒的复制和蛋白表达。HSV间层蛋白UL13和US3是Ser/Thr蛋白激酶。与PKA类似，US3具有磷酸化CREB的能力，而UL13是US3上游激酶。据此，我们推断疱疹病毒的Ser/Thr蛋白激酶可通过CREB-BZLF1通路激活潜伏感染的EBV。本课题将围绕这一假说，通过构建HSV US3/UL13缺失病毒等方法，发现疱疹病毒激活潜伏感染EBV的分子机制，为临床诊治提供依据。

潜伏感染是Epstein-Barr病毒（EBV）等疱疹病毒的特征。EBV可引起鼻咽癌和淋巴瘤等重大疾病，潜伏感染的激活与其致病机制密切相关。我们前期研究发现，HSV的共感染可激活潜伏感染的EBV（已发表）。本项目围绕着疱疹病毒激活潜伏感染EBV的分子机制，从四个层次展开研究，取得以下结论：1）疱疹病毒的Ser/Thr蛋白激酶UL13不激活潜伏感染的EBV；2) HSV感染通过内质网应激IRE1-XBP1通路激活潜伏感染的EBV；3)鱼腥草水提物及其化学组分Houttuynoid A具有抗HSV-1病毒活性；4) 系统研究EBV编码蛋白的亚细胞定位，发现其定位与功能密切相关，亦研究HSV编码蛋白UL2的亚细胞定位和定位机制。本项目回答了一系列基本的科学问题，做出了原创性的贡献，也将疱疹病毒的抗病毒药物扩展到动物模型，具有潜在的临床应用。