Prediction Of Mirna Encoded By Genome Of CGSIV And Structural Analysis And Functional Characterization Of MD272

Micro RNAs(mi RNAs) are key regulators of gene expression in higher eukaryotes. Recently, a large number of mi RNAs have been identified for DNA viruses such as herpesviruses, polyomaviruses, ascoviruses and adenoviruses, which have evolved to exploit RNA silencing for regulating the expression of their own genes, host genes, or both.In the past years, a kind of DNA virus was isolated from diseased farmed Chinese giant salamanders(Andrias davidianus) at different areas in China. It was the Chinese giant salamander virus(CGSV)/ Andrias davidianus iridovirus(ADIV). In 2011, the virus(designated as Chinese giant salamander iridovirus; CGSIV) was isolated from the liver and kidney of the diseased salamanders from in Zhangjiajie National Forest Park, Hunan Province, China. CGSIV causes serious systemic diseases and high mortality rates in farmed Chinese giant salamanders,and was identified as a member of the CMTV group in amphibian-like ranavirus(ALRV) of Ranavirus genus of Iridoviridae family.In the previous studies, functional genomics analysis of Chinese Giant Salamander Iridovirus was carried out. We have determined the complete genome sequence and virion proteome of the CGSIV. The genome is 10,6366 bp, has a G+C content of 55%. A total of 112 putative open reading frames, including all of the iridovirus- and ranavirus-specific genes as well ALRV-specific genes, were identified by RT-PCR.To aid in the understanding of the molecular mechanisms of CGSIV,we performed prediction and functional analysis of mi RNA encoded by genome of the CGSIV.In this study, combined analyses for pre-mi RNA sequences using VMir and Mi Pred softwares generated 19 possible pre-mi RNAs in CGSIV genome. The secondary structures of mi RNA precursor sequences were predicted by RNAfold software. In CGSIV mi RNA precursors, fourteen were located within ORFs with known functions or predicted motifs. Nine out of 14 CGSIV predicted mi RNAs were located in opposite strands to the coding genes. Thus, it is possible that the potential mi RNAs encoded by CGSIV regulate the expression of its own genes that play important roles in virus replication and pathogenesis. However, how these mi RNAs function needs further investigation.One of them, CGSIV MD272(mi R-MCP) was found to lie in the opposite transcriptional orientation of ORF019 L. CGSIV-ORF019 L encodes the major capsid protein(MCP) for viral particle. MCP is an essential virion protein and a single polypeptide with a molecular weight of about 50 k Da, accounting for up to 45% of all viral proteins and representing 90% of the proteins solubilized from the virions. CGSIV MD272 may regulate the expression of MCP. To confirm MD272 transcriptional expression, we performed RT-PCR on total RNA extracted from the cultures using primer pairs specific for EPC 18 S r RNA and MD272. The transcript of MD272 was detected by RT-PCR after 4 h of CGSIV infection under mock infection control.Major capsid proteins are involved in the assembly of viral particles, and a reduction in the MCP expression levels correlates with a reduction in production of infectious new particles. In an attempt to further confirm the effect of gene silencing by mi RNA against MCP we assayed the si RNA(mi R-MCP) as MD272. As shown in this report, mi R-MCP- treatment had the effect on replication at MOIs of 0.5 PFU/cell in plaque assay. The resulting virus yields from the culture infected with anti-MCP-si RNA were reduced to 70% at 24 h.p.i and 50% at 48 h.p.i in control treated cultures. The results were determined for MCP expression by si RNA-mediated silencing with Western blot analysis.In this report, mi RNA precursors of CGSIV were predicted. One of the precursors may regulate the MCP expression. si RNA as the MD272 sequence was designed and silenced MCP expression. The results provide a useful resource for further in-depth studies on mi RNA role in viral infection.