Cloning And Characterization Of Three Immune Evasion Genes From Iridovirus

Lymphocystis disease virus isolated from China (LCDV-C) and Rana grylio virus (RGV) were important virus agents isolated from the diseased flounder and Rana grylio in China, respectively. Based on the sequencing and annotation of the LCDV-C genome, and the studies on the morphogenesis and physicochemical characterizations of RGV, we cloned and detected the functions of three immune evasion genes from LCDV-C, and investigated the mechanism of RGV induced apoptosis in fish cells. The results are given as follows:1. The reannotation of open reading frame (ORF) from LCDV-C indicated that at least three immune evasion gene homologs were present in LCDV-C, including G protein coupled receptor (GPCR) (ORF124R), tumor necrosis factor receptor (TNFR) (ORF095R) and Bcl-2 (ORF189R). The characterization of these genes was described as following.(1). The transmembrane prediction of LCDV-C GPCR indicated that it had a central core domain constituted of seven transmembrane regions and a highly conserved DRY motif. The homology search showed that LCDV-C GPCR shared 37% identity to flounder chemokine receptor 9 (CCR9), following by rainbow trout CXCR, zebrafish CXCR4 and other mammal virus. Full length of LCDV-C GPCR was obtained by PCR amplification using LCDV-C DNA as template. The recombinant plasmid GPCR-GFP and pcDNA-GPCR were constructed and confirmed by sequencing, and the subcellular localizatization and function in fish cells were detected. The results showed that: LCDV-C GPCR was expressed in both the cytoplasm and nucleoplasm of fish cells and independent of the cell type; No obvious cytotoxic effect was examined after transient overexpression of LCDV-C GPCR in fish cells; Stabled expressed LCDV-C GPCR not only increased the cell proliferation, but also had the transformation potential in FHM cells. In addition, LCDV-C GPCR overexpression also inhibited apoptosis under external apoptotic stimuli, such as CHX and ActD. Considering Lymphocystis is a chronic disease characterized by wart-like lesions, the transforming potential of LCDV-C GPCR, which maybe functioned via inhibiting apoptosis, was purposed to contribute to the massive enlargement of epidermal cells and viral persistent infection.(2). The computer assisted analysis on LCDV-C Bcl-2 showed that it shared 34% identity to zebrafish Mcl and had a transmembrane domain at C terminal. The detection of subcellular localization of LCDV-C Bcl-2 by using GFP fusion protein showed that LCDV-C colocalised to endoplasmic reticulum (ER), while its transmembrane mutant had a changed localization. These data indicated that the transmembrane domain is important for LCDV-C Bcl-2 localization. The transient expression of LCDV-C Bcl-2 had no obvious cytotoxic effect on fish cells, but the overexpression of LCDV-C Bcl-2 increased the apoptosis induced by ActD and the deletion of transmembrane domain influenced the ability of enhancing apoptosis. In addition, LCDV-C Bcl-2 expression inhibited the NF-κB activation induced by ActD.(3). The domain analysis of LCDV-C TNFR showed that it had the conserved cysteine rich domain which is a typical chacterization of TNFR family members. Although it shared 38% identity to mouse TNFR2, the identity between LCDV-C TNFR and LCDV-1 or LCDV-2 TNFR was less than 20%. It is indicated that the difference of gene structure existed between various iridovirus isolates. The examination of LCDV-C TNFR localization showed that LCDV-C TNFR was also colocalized to ER, this data is different from the subcellular localization of other viral TNFRs. Transient expression LCDV-TNFR could not induce apoptosis in fish cells, but the stably overexpression of LCDV-C TNFR enhanced the apoptosis induced by ActD and CHX, and inhibited the ActD activated NF-κB.2. Using electron micrscopy and fluorescent microscopy observation, reporter gene assay, examination on the intracellular Ca2+ and caspase activity, we investigate the mechanism of RGV induced apoptosis in fish cells. The data showed that RGV infection induced a series of changes, such as mitochondrial fragmentation, caspase activation, NF-κB and AP-1 activation and intracellular Ca2+ increase. Combining the previous results that RGV infection altered the mitochondrial distribution and the mitochondrial membrane potential (Δψm) collapse, we purposed that RGV infection should trigger mitochondrion mediated apoptosis.Taken together, the results not only provided important clues for the studies on iridovirus immune evasion and the virus-host interaction at molecular level, but also contributed to understanding the iridovirus pathogenesis.