Functional analysis of viral protein kinases and regulatory proteins for latency of herpes simples virus

单纯疱疹病毒（HSV）是人类重要的病原体。在本研究中，我们重点研究了两个病毒调节蛋白ICP0和UL13。此外，我们试图通过分析lCP0和UL13的功能同源物来揭示这些HSV调节蛋白的新特征。所得结果如下：通过酵母双杂交筛选，我们确定了碱性螺旋-环-螺旋（bHLH） PAS超家族成员BMAL1作为新的ICP0结合伙伴。我们发现ICP0与BMAL1相互作用并刺激BMAL1依赖性转录。此外，我们证实了ICP0参与了感染细胞细胞质中BMAL1的稳定。bHLH-PAS超家族成员是应激相关蛋白，通常在适应环境变化中发挥作用。HSV在应激条件下被重新激活，据报道，ICP0调节潜伏期的重新激活。综上所述，我们的研究结果表明，ICP0和BMAL1之间的相互作用可能在病毒反应中发挥作用。eb病毒也属于疱疹病毒科。我们重点研究EBV编码的蛋白激酶BGLF4，这是HSV UL13的同源物。我们成功建立了用重组杆状病毒表达和纯化GST和his标记的BGLF4的系统。我们还发现BGLF4的一个细胞靶点是翻译因子EF- 1δ。我们之前发现HSV UL13也磷酸化EF-1δ。综上所述，这些结果表明EF- 1δ在感染细胞中是所有疱疹病毒的保守功能，EF- 1δ在各种疱疹病毒感染中起重要作用。我们还关注了另一种EBV调控蛋白ena - lp，该蛋白被认为是HSV ICP0的功能同源物。我们的研究结果表明，核基质结合、核定位和细胞激酶磷酸化是表达ena - lp共激活因子功能的必要条件。ICP0也可能被细胞激酶磷酸化，并定位于核基质中。这些结果表明，细胞激酶对ICP0的定位和磷酸化也是表达ICP0功能的关键。少

Herpes simplex virus (HSV) is an important pathogen in human. In this research we focused on two viral regulatory proteins ICP0 and UL13. Furthermore, we tried to unveil novel features of these HSV regulatory proteins by analyzing functional homologs of lCP0 and UL13. The results obtained were as follows.By yeast two-hybrid screening, we identified BMAL1, a member of basic helix-loop-helix (bHLH) PAS super family, as a new ICP0 binding partner. We found that ICP0 interacts with BMAL1 and stimulates BMAL1 dependent transcription. Furthermore, we demonstrated that ICP0 is involved in stabilization of BMAL1 in the cytoplasms of infected cells. Members of bHLH-PAS super-family are stress-related proteins and often play roles in adaptation to environmental changes. HSV is reactivated under stress conditions and it has been reported that ICP0 regulates reactivation from latency. Taken together, our findings suggest that the interaction between ICP0 and BMAL1 may play a role in viral reactiva … More tion from latency.Epstein-Barr virus (EBV) also belongs to family Herpesviridae. We focused on EBV encoded protein kinase BGLF4 which is a homolog of HSV UL13. We succeeded to establish system for expression and purification of GST- and His-tagged BGLF4 using recombinant baculovirus. We also found that a cellular target of BGLF4 is translation factor EF- 1δ. We previously showed that HSV UL13 also phosphorylate EF-1δ. Taken together these results indicate that phosphorylation of EF- 1δ in infected cells is conserved function of all herpesviruses and EF- 1δ plays important role in various herpesvirus infections. We also focused on another EBV regulatory protein EBNA-LP which is suggested to be a functional homolog of HSV ICP0. Our results indicated that nuclear matrix association, nuclear localization, and phosphorylation by cellualr kinase(s) is essential to express co-activator function of EBNA-LP. ICP0 is also suggested to be phosphorylated by cellular kinase(s) and to be localized in nuclear matrix. These results suggest that cellular localization and phosphorylaton of ICP0 by cellular kinase(s) is also critical to express ICP0 functions. Less