禽网状内皮组织增生病病毒与宿主细胞波形蛋白相互作用机制的研究

Reticuloendotheliosis (RE) is an acute reticulum cell tumor, lymph and other tissue chronic tumor, runting-stunting syndrome of multiple avian species that is caused by reticuloendotheliosis virus (REV), REV genome consists of three structural genes (gag, pol and env) flanked by long-terminal repeats (LTRs), the pol gene encodes the enzyme protein: protease, reverse transcriptase and integrase enzyme. The previous research on the differential proteomic analysis of the spleen of SPF chicken infected with REV showed that the expressions of cytoskeletal protein Vimentin were down-regulated at different time post REV infection. However, the exact mechanism is unclear. This study aims to investigate the mechanism of the interaction between REV and host cell vimentin. In this program, a series of REV pol protein eukaryotic expression plasmid, including the truncated as well as amino acid mutations, were constructed and transfected into the chick embryo fibroblast cells to determine the core area and the critical amino acid sites of REV pol protein that affect vimentin expression. Furthermore, the vimentin overexpression cell lines and the vimentin silence-expression cell lines were constructed and identified, in order to analyze the biological functions of vimentin played in REV replication. The study will help to well understand the mechanism of the interaction of REV with host cell vimentin, and provide a theoretical basis for the understanding of REV infection and its pathogenesis.

网状内皮组织增生病(RE)是由网状内皮组织增生病毒(REV)引起的一类以矮小综合征、禽类急性网状细胞肿瘤、淋巴以及其他组织的慢性肿瘤为特征的病理综合征。REV基因组由gag、pol、env等基因组成，其中pol基因主要编码酶蛋白：蛋白酶、反转录酶和整合酶。本课题组对REV感染SPF鸡脾脏差异蛋白质组学研究发现，细胞骨架蛋白Vimentin在病毒感染后的不同时间点均呈现不同程度的下调表达。但其确切机制目前尚不清楚。本项目通过构建一系列REV pol蛋白截短表达以及氨基酸突变的真核表达质粒转染细胞，确定REV pol蛋白影响vimentin表达的核心区域及关键的氨基酸位点；并通过构建并筛选vimentin过表达细胞系和vimentin沉默表达细胞系，分析vimentin在REV复制中发挥的生物学功能，阐明REV与宿主细胞vimentin相互作用的机制，为了解REV感染及其发病机制提供理论基础。

网状内皮组织增生病(RE)是由网状内皮组织增生病毒(REV)引起的一类以矮小综合征、禽类急性网状细胞肿瘤、淋巴以及其他组织的慢性肿瘤为特征的病理综合征。REV基因组由gag、pol、env等基因组成，其中pol基因主要编码酶蛋白：蛋白酶、反转录酶和整合酶。本课题组对REV感染SPF鸡脾脏差异蛋白质组学研究发现，细胞骨架蛋白Vimentin在病毒感染后的不同时间点均呈现不同程度的下调表达。但其确切机制目前尚不清楚。.本项目通过构建密码子优化的REV PR蛋白的原核、真核表达质粒，证明了REV PR在体内、体外对Vimentin均具有切割作用，且这一活性的发挥依赖于PR蛋白的完整结构。此外，PR蛋白DTG活性序列的突变、PR抑制剂的存在、以及反应体系的pH值和钠离子的浓度均影响PR蛋白对Vimentin的切割活性；激光共聚焦结果显示：转染PR真核质粒的细胞中vimentin蛋白由均匀分布状态变得聚集。通过利用RNA干扰和vimentin抑制剂的方式下调表达细胞中vimentin，发现REV在vimentin下调表达的细胞中的复制受到了明显的抑制。.本项目通过上述研究内容，阐明了REV与宿主细胞vimentin相互作用的机制，为了解REV感染及其发病机制提供理论基础。