单链正义RNA病毒的复制往往发生在细胞内特殊的膜结构上，而病毒复制酶小亚基在介导膜结构的改变以及复制"工厂"的形成中发挥着关键作用。在前期工作的基础上，本项目拟：(1) 利用细胞生物学等手段明确甜菜黑色焦枯病毒（BBSV）复制酶小亚基p23的亚细胞定位，确定其关键跨膜区；(2) 通过观察BBSV基因组RNA的定位以及p23所致细胞内膜结构的改变，明确BBSV在细胞内复制的场所；(3) 分析p23与寄主Hsp70和Hsp90的相互作用在BBSV侵染过程中的作用；(4) 分析p23超表达导致叶片坏死的机制，重点研究蛋白错误折叠反应（UPR）在p23的致病以及BBSV复制增殖过程中的作用。通过以上工作明确BBSV复制酶小亚基p23的亚细胞定位及其在病毒致病过程中的作用机制，提出坏死病毒属（Necrovirus）病毒的复制模型，丰富人们对番茄丛矮病毒科（Tombusviridae）的认识。

The replication of positive single strand RNA plant viruses depends on specific endomembrane, and the auxiliary replicase protein play a pivotal role in the morphological alterations of endomembranes and the formation of viral replication factory. Based upon previous studies, in this proposal, (1) we will use the methods of cell biology to analyze the subcellular localization of the p23 auxiliary replicase protein of Beet black scorch virus, the critical domain that determine the membrane localization of p23 will also be mapped. (2) Meanwhile, the pathological morphology alteration of the endomembrane during the expression of p23 as well as the subcellular localization of BBSV genomic RNA will also be investigated, and then to ascertain the site for the assembly of virus replication complex (VRC). (3) Previous BiFC assay showed that p23 could interact with Hsp70 and Hsp90 in vivo, respectively. Next, further experiments will be conducted to confirm this result, and the roles of Hsp70 and Hsp90 involved in the infection of BBSV will be systemically studied. (4) Overexpression of p23 could induce cell death in the inoculated leaves and preliminary results indicated that this phenomenon was probably related with the activation of unfolded protein response (UPR). We will further study the roles of UPR involved in the pathogenicity of the p23 as well as in BBSV infection, and finally a model for the replication of Necrovirus will be proposed. Our results will broaden the current understanding of the replication model for the virus in the Tombusviridae.