|Abstract||虹彩病毒是危害水产养殖动物的重要病毒性病原之一，给世界各地淡、海水养殖业造成了严重的经济损失。蛙虹彩病毒（Rana grylio virus, RGV）和淋巴囊肿病毒中国分离株（Lymphocystis disease virus isolated from China, LCDV-C）是本实验室分别从患病的养殖沼泽绿牛蛙和牙鲆中分离得到的两种虹彩病毒。本论文以RGV和LCDV-C为研究对象，分别鉴定四个重要的功能基因，其中三个核酸代谢相关基因和一个病毒结构基因，并对其表达特征和功能进行了研究，主要结果如下：
1、 蛙虹彩病毒尿嘧啶脱氧核糖核苷三磷酸酶（RGV deoxyuridine triphosphatase, RGV dUTPase）基因：ORF 全长495bp，编码长为164aa、分子量约为17.4 kDa的蛋白。序列比对显示RGV dUTPase含有与细胞dUTPase酶活性所必需的5个保守结构域和21个不变的氨基酸残基。5′-RACE分析表明RGV dUTPase基因的转录起始位点位于翻译起始密码子ATG上游第15个核苷酸处。RT-PCR和Western blotting分析显示，RGV dUTPase基因在病毒感染细胞后4h开始转录和表达，随着感染时间延长表达量逐渐增加。放线菌酮CHX和阿糖胞苷AraC抑制实验表明，RGV dUTPase基因属于早期表达的基因。通过GFP融合蛋白瞬时表达和免疫荧光对其亚细胞定位进行分析，显示在鱼类EPC细胞内RGV dUTPase是专一细胞质定位蛋白。
2、 牙鲆淋巴囊肿病毒胸苷酸合酶（LCDV-C thymidylate synthase, LCDV-C TS）基因： ORF 全长858bp，编码长为286aa、分子量为32.7 kDa的蛋白。序列比对显示LCDV-C TS含有与细胞TS酶活性所必需的24个保守氨基酸残基和4个功能结构域。亚细胞定位显示，LCDV-C TS在鱼类细胞中定位在细胞质中，并呈颗粒状分布。利用流式细胞仪对细胞周期分析，表明组成性表达LCDV-C TS导致细胞周期的时相比发生变化，其中S期、G2/M期细胞比率上升，从而使细胞生长速度增快。细胞集落实验显示LCDV-C TS组成性表达使细胞失去了接触抑制能力，堆积生长，形成岛屿状细胞集落；软琼脂培养实验进一步证明这些细胞已经获得了定着非依赖生长表型，能在0.3% 的软琼脂中生长、形成细胞克隆。在血清饥饿的情况下，LCDV-C TS组成性表达诱导细胞凋亡。可见，LCDV-C TS的组成性表达诱导了鱼类细胞产生转化表型；这是病毒TS基因具有转化细胞潜能的首次报道。
3、 蛙虹彩病毒胸苷激酶（RGV thymidine kinase, RGV TK）基因：ORF 全长588bp，编码长为195aa、分子量为22.1kDa的蛋白。序列比对和系统进化分析显示RGV TK含有保守的核苷酸结合结构域，与细胞的核苷酸激酶（deoxyribonucleoside kinase, dNK）家族中TK2, dCK和dGK亲缘关系较近。Western blotting分析显示，RGV TK基因在病毒感染细胞后6h开始表达，随着感染时间延长蛋白表达量不断增加。CHX和AraC抑制实验表明，该基因属于早期表达的基因。通过GFP融合蛋白瞬时表达和免疫荧光对其进行亚细胞定位，显示RGV TK在鱼类FHM和GCO细胞中是专一细胞质定位蛋白。利用RNAi技术敲降了RGV TK基因的表达，但病毒滴度并没有明显的下降；这表明TK基因对RGV在鱼类培养细胞中的复制不是必需的。
4、 蛙虹彩病毒囊膜蛋白（RGV envelope protein 53R, RGV 53R）基因：ORF 全长1569bp，编码长为522aa、分子量为54.7 kDa的蛋白。RGV 53R是虹彩病毒核心基因，具有病毒囊膜蛋白的结构特征：跨膜结构域，十四烷基化位点和保守的半胱氨酸残基。RT-PCR和Western blotting分析显示，RGV 53R基因在病毒感染细胞后12h才开始转录和表达，且能被AraC所抑制，这表明它属于晚期表达的基因。免疫荧光定位显示在病毒感染早期53R蛋白在细胞质中呈颗粒状分布，而晚期则定位于病毒加工厂。53R蛋白的这种动态分布在53R-GFP融合蛋白表达实验中也得到证实：在转染早期，53R-GFP蛋白定位于内质网上，但是在转染晚期53R-GFP蛋白发生聚集，并与病毒加工厂共定位。另外，Western blotting显示53R蛋白存在RGV病毒的囊膜上；抗体中和实验显示53R抗体能明显降低或延迟RGV的感染。可见， RGV 53R是一个新的虹彩病毒囊膜蛋白，在病毒的装配和感染过程中发挥重要作用。|
|Other Abstract||Iridoviruses are one of significant viral pathogens for aquatic animals which have caused great economic losses in aquaculture industry worldwide. Rana grylio virus (RGV) and Lymphocystis disease virus – China (LCDV-C) were respectively isolated from the diseased Rana grylio and Paralichthys olivaceus in China and had been identified as two members of the family Iridoviridae. In this study, we cloned four functional genes from RGV and LCDV-C genomes, and subsequently characterized their temporal expression pattern, subcellular localization and possible function. The results are given as follows:
1. RGV deoxyuridine triphosphatase (RGV dUTPase) gene: RGV dUTPase encodes a protein of 164aa with a predicted molecular mass of 17.4 kDa. Multiple sequences alignment showed that RGV dUTPase was quite similar to cellular dUTPases. Subsequently, RT-PCR and Western blotting detection revealed RGV dUTPase was transcribed and translated as early as 4 h postinfection (p.i.), and its transcriptional initiation site was determined by 5′RACE to start from the nucleotide A at 15 nt upstream of the initiation codon ATG. Using drug inhibition analysis by a de novo protein synthesis inhibitor (cycloheximide, CHX) and a viral DNA replication inhibitor (cytosine arabinofuranoside, AraC), RGV dUTPase was classified as an early (E) viral gene during the in vitro infection. Subcellular localization of RGV dUTPase was investigated using dUTPase-GFP fusion protein and immunofluorescence staining, and revealed that it was nearly exclusively a cytoplasmic protein, but not presented into virus factory. Moreover, constitutive expression of RGV dUTPase was shown that there was no effect on RGV replication by viral replication kinetics assay.
2. LCDV-C thymidylate synthase (LCDV-C TS) gene: LCDV-C TS encodes a protein of 286aa with a predicted molecular mass of 32.7 kDa. Multiple sequences alignment revealed that it was quite similar to other identified TS and contains four conserved functional motifs. Subcellular localization revealed that LCDV-C TS was predominantly localized in the cytoplasm with granular appearance in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS expressed cells gave rise to foci 4–5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage independent phenotype and could grow in 0.3% soft agar. In addition, we show that constitutive expression of LCDV-C TS results in apoptotic cell death following serum removal. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity.
3. RGV thymidine kinase (RGV TK) gene: RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologs of the protein are present in all currently sequenced iridoviruses, and database searches found it appeared more closely related to cellular TK type 2 (TK2) and deoxycytidine kinases (dCKs). Subsequently, Western blotting and drug inhibition analysis revealed TK expression increased with time from 6 h post-infection in RGV infected-cells and RGV TK belongs to the early expression class of genes during the in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was nearly exclusively a cytoplasmic protein in fish cells, but excluded from the virus factory. Moreover, knockdown of RGV TK expression by RNAi technique was shown that there was no effect on virus yields of RGV and synthesis of late viral proteins in GCO cells. Collectively, the current studies indicate that RGV TK, an early gene of iridovirus, encodes a cytoplasmic protein which might be not essential for virus replication in cultured fish cells.
4. RGV envelope protein 53R (RGV 53R) gene: RGV 53R encodes a protein of 522aa with a predicted molecular mass of 54.7 kDa. Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed several conserved structural features shared by virus capsid or envelope proteins: a myristoylation site, two predicted transmembrane domains and two invariant cysteine residues. Subsequently, RT-PCR and Western blotting detection revealed that the transcripts encoding RGV 53R and the protein itself appeared late during infection of fathead minnow cells and that their appearance was blocked by viral DNA replication inhibitor, indicating that RGV 53R is a late expression gene. Moreover, immunofluorescence localization found association of 53R with virus factories in RGV-infected cells, and this association was further confirmed by expressing a 53R–GFP fusion protein in pEGFP/53R-transfected cells. Detergent extraction and Western blotting detection confirmed that RGV 53R was associated with virion membrane. Furthermore, Antibody neutralization assay revealed that RGV infection is significantly inhibited by anti-53R serum. Therefore, the current data suggest that RGV 53R is a novel viral envelope protein and that may play an important role in virus assembly and infection. This is thought to be the first report on a viral envelope protein that is conserved in all sequenced iridoviruses.|