Rep/RepA对香蕉束顶病毒复制的作用机制研究

Banana is one of the main crops in tropical and subtropical regions. The pathogen of Banana bunchy top virus (BBTV) is a major disease in the main banana-producing areas of China. BBTV is a multi-component, single-stranded DNA virus. Most of the BBTV isolates in China contain two DNA components that encode Rep-like proteins that direct BBTV DNA replication. The proteins of Rep encode by DNA1 and RepA encoded by α-satellite DNA have the ability to initiate BBTV DNA replication. Rep has been reported to direct its own replication and replication of DNA3 and DNA 5 while RepA directed its own replication. However, the template specificity of Rep/RepA remains unresolved. Transcriptomic analysis revealed that α-satellite DNA along with DNA6 is highly expressed and maybe is more abundant. It is possible that RepA could direct the replication of DNA6 in addition to its own DNA. To study whether Rep and RepA direct self-replication or replication of other BBTV components, we propose to construct BBTV infectious clones and use these clones to study self-directed or trans-directed replication. Infectious DNA clones will be bombarded into banana suspension cells by the gene gun method alone or in combination with other DNA components. Mutant DNA1 and α-satellite DNA will also be constructed to test if they direct replication of each other’s DNA. To understand the molecular mechanism of these templates specificity, Rep and RepA binding sites will be identified by gel shift assay and DNase I footprinting experiments. Selected site-directed mutations will be carried out to verify the DNA binding site and the template specificity. The results of this project will eventually resolve the molecular mechanisms of template specificity for Rep and RepA proteins. Completion of this study will provide insights into the early stage of DNA replication in BBTV infection and a theoretical basis for the study of anti-BBTV pathway.

香蕉束顶病毒(Banana bunchy top virus, BBTV)是香蕉上危害最严重的病毒之一。基因组由6~8个大小约1kb的环状ssDNA组成，其中DNA1和卫星组分分别编码Rep和RepA蛋白，均参与BBTV复制。Rep参与病毒DNA1、DNA3及DNA5组分的复制，而RepA参与卫星组分的自我复制。申请者前期发现，这两种复制相关蛋白的上述差异，可能与DNA1和卫星组分茎环共同区核苷酸序列的不同有关。本项目拟对Rep和RepA在BBTV的复制中所起的不同作用及其分子机制展开研究，试图通过侵染性克隆等试验方法找到二者不同复制调节作用的分子基础，并通过点突变实验来验证这一理论假设，从而揭示BBTV感染香蕉早期Rep和RepA对病毒基因组的复制所起的不同作用和特异结合位点。本项目将较系统地阐明BBTV的DNA复制过程，为探索防治香蕉束顶病毒的手段提供理论基础。

基于滚环复制，香蕉束顶病毒Rep/RepA对病毒环状单链DNA进行剪切和连接。本项目将BBTV基因组的8个环状单链DNA片段分别构建到植物双向表达载体pCAMBIA3300 中，以农杆菌介导转化野生型烟草和巴西蕉叶片，反向PCR和病症结果显示，成功构建了BBTV侵染性克隆且能感染香蕉宿主。将BBTV各组分重组质粒的农杆菌单独注射或共同注射烟草，反向PCR结果表明DNA-R编码的Rep蛋白能对自身组分及5个其他必要组分进行剪切和连接，产生子代环状DNA；而卫星组分（NewS2、Sat4）仅能对自身组分进行剪切和连接。对感染BBTV后期的香蕉样品各组分进行实时荧光定量PCR，结果表明，该样品叶片中各组分DNA比例关系为DNA-R: DNA-U3: DNA-S: DNA-M: DNA-C: DNA-N=2: 12 : 3: 8: 1: 9。说明在宿主复制酶和病毒Rep蛋白的共同作用下，产生的DNA-U3、DNA-M和DNA-N组分DNA含量较多，而产生的DNA-R、DNA-S和DNA-C的DNA 含量相对较少。对DNA-R、DNA-U3、DNA-S、DNA-M、DNA-C和DNA-N组分的非编码基因序列进行生物学分析，结果表明BBTV各组分非编码区都含有一个9 bp（TATTATTAC）的茎环结构区（CR-SL）。与Nanovirus 病毒和双生病毒相似，该区域是Rep蛋白结合的保守区域，突变其位点影响Rep蛋白对环状组分的剪切和连接效率。综上所述，本研究揭示了BBTV Rep和RepA对病毒基因组复制所起的不同作用和特异结合位点，较系统地阐明BBTV的DNA复制过程，为探索防治香蕉束顶病的手段提供理论基础。