原癌蛋白c-Myc与鲤疱疹病毒Ⅱ型立即早期蛋白ORF121互作调控病毒复制机理

鲤疱疹病毒Ⅱ型(CyHV-2)感染鲫或金鱼导致造血器官坏死病，该病具有极高的死亡率给鲫养殖业造成了巨大的经济损失。疱疹病毒立即早期蛋白在病毒复制过程中起着重要的作用，但尚不清楚其在鱼类疱疹病毒感染中如何发挥作用。前期利用酵母双杂交筛选鲫cDNA文库，发现CyHV-2立即早期蛋白ORF121与鲫c-Myc互作，Far-Western证实两者体外结合。为阐明ORF121与c-Myc互作介导c-Myc及其相关信号通路调控病毒复制的分子机理。本研究拟进行c-Myc的敲降和过表达，研究c-Myc对病毒复制的影响；通过Co-IP和共聚焦等技术解析ORF121和c-Myc作用的关键结构域，研究二者互作对病毒复制的作用；利用双荧光素酶报告基因系统研究病毒立即早期蛋白ORF121介导c-Myc相关信号通路调控病毒复制分子路径。为揭示鱼类疱疹病毒立即早期蛋白调控宿主介导其复制的分子机制提供重要的理论基础。

Cyprinid herpesvirus 2(CyHV-2) is the causative agent of herpesviral hematopoietic necrosis(HVHN) disease in crucian carp and gold fish. HVHN causes significant losses in crucian carp aquaculture with high mortality. Herpesvirus encodes immediate early proteins(IE protein) which are usually key players during the viral infection. But the role of the fish herpesvirus immediate early protein in invasion is unclear. Through yeast two-hybrid system screen with the crucian carp cDNA library, cellular myelocytomatosis oncogene(c-Myc) was identified as a potential interacting partner for the immediate early protein ORF121 of CyHV-2. Far-western blot assay also demonstrated that ORF121 interacted with c-Myc in vitro. The purpose of this proposal is to clarify the molecular mechanism of CyHV-2 infection mediated by the interaction between viral ORF121 and cellular c-Myc and its downstream signaling pathways. In this proposal, we plan to investigate the effects of c-Myc on the viral infection with c-Myc knockdown or overexpression. Meanwhile, Co-IP and confocal microscopy system would be performed to investigate the effect of protein-protein interaction on virus invasion through analysing the key domains between ORF121 and c-Myc. Furthermore, luciferase system would be performed to investigate the molecular mechanism of CyHV-2 infection mediated through the regulation of downstream signaling pathway of the cellular c-Myc by viral ORF121. Our proposal would help us understand the mechanism of fish herpesvirus infection mediated by the viral immediate early protein.