Molecular Cloning And Characterization Of Two Novel Viral Genes, 3β-HSD And PCNA, From Rana Grylio Virus

Rana grylio virus (RGV), a member of Ranavirus (family Iridoviridae), was isolated from diseased pig frog Rana grylio. This study is attempting to explore the functional genes from RGV, and then provide some fundamental knowledge for the control of RGV infection. Two novel viral genes were cloned from RGV using conserved oligonucleotide primers, and then several experiments have been carried out to identify their characterization. The main research results are as following.1. Characterization of RGV 3β-hydroxysteroid dehydrogenase (3β-HSD) gene and functional analyses of RGV 3β-HSD overexpression.(1) The full length of RGV 3β-HSD ORF has 1068bp, encoding a 355aa deduced protein with a theoretical molecular weight of 39.3 KDa. Upon the homology search and amino acid alignment, the deduced amino acid sequence of RGV 3β-HSD shows the highest identity of 47% with frog (Xenopus laevis) 3β-HSD. Also, it was revealed that the deduced amino acid sequence of RGV 3β-HSD contains some conserved amino acids or motifs that are critical for the function of 3β-HSD.(2) By RT-PCR analysis, it was revealed that the transcript of RGV 3β-HSD could be detected at 4h post-infection (h p.i.) and continued to be present at high level during 4-48 h post-infection (h p.i.). By rapid amplification of 5'-terminal cDNA end (5'-RACE), the transcription initiation site of RGV 3β-HSD was identified to located at 19 nucleotides (nt) upstream of the translation start site. Furthermore, cycloheximide (CHX), a de novo protein synthesis inhibitor, and cytosine arbinoside (AraC), a DNA synthesis inhibitor, were utilized to classify the transcript of RGV 3β-HSD gene. The result showed that RGV 3β-HSD should be an immediate-early (IE) viral gene.(3) Upon eukaryotic transfection and fluorescence microscopy observation, the RGV 3β-HSD with a C-terminal enhanced green fluorescent protein (EGFP) tag was revealed to locate in the cytoplasm of the transfected cells, including epithelioma papulosum cyprini (EPC), fathead minnow (FHM), and baby hamster kidney (BHK-21). To further evaluate the subcellular localization of RGV 3β-HSD, two organelle-specific markers, pDsRed2-ER (endoplasmic reticulum) and pDsRed2-Mito (mitochondia), were used. Observation of the cotransfected EPC cells by confocal microscopy showed that RGV 3β-HSD was exclusively associated with cellular mitochondria, suggesting that the subcellular localization of a virus-encoded 3β-HSD is quite different from that of vertebrate 3β-HSD.(4) EPC cells were trasfected with pcDNA3.1-HSD and empty vector pcDNA3.1, selected by G418, and confirmed by RT-PCR to obtain two stable transfectants, EPC/pcDNA3.1-HSD and EPC/pcDNA3.1. Upon the morphological observation and MTT quantitative analyses, it was found that overexpression of RGV 3β-HSD could significantly inhibit the cell death induced by either RGV or Scophthalmus Maximus Rhabdovirus (SMRV). Furthermore, the results of DNA agarose gel electrophoresis and flow cytometry revealed that overexpression of RGV 3β-HSD could significantly inhibit SMRV-induced apoptosis. All these suggest that RGV 3β-HSD might be a novel anti-apoptosis factor.2. Characterization of RGV proliferating cell nuclear antigen (PCNA) gene.(1) The full length of RGV PCNA ORF was identified to be 1068bp, encoding a 245aa deduced protein with a theoretical molecular weight of 26KDa. The deduced amino acid sequence of RGV PCNA shows poor identity with eukaryotic PCNA, and has the highest identity of only 18% with frog (Xenopus laevis) PCNA. However, the secondary structure of RGV PCNA was revealed to have striking similarity with that of eukaryotic PCNA, and contain the conserved domains that are critical for the function of PCNA.(2) A prokaryotic expression recombinant plasmid, pET32a-PCNA, was constructed and introduced into the E. coli. strain BL21(DE3) for expression. The fusion protein band was detected by SDS-PAGE and purified by Ni-IDA His·Bind affinity column. The purified fusion protein was utilized to immunize mouse, and then mouse anti-PCNA serum was prepared.(3) By RT-PCR and western blot analyses, the expression of RGV PCNA could be detected as early as 4h p.i. and continued to be present during the infection at both RNA level and protein level. CHX and AraC inhibition assay showed that RGV PCNA should be a late (L) viral gene.(4) By eukaryotic transfection and fluorescence microscopy, it was found that RGV PCNA with a C-terminal EGFP tag is aggregated in the cytoplasm of the transfected cells of EPC, FHM, and BHK-21. Moreover, using immunofluorescence assay on RGV-infected FHM cells, it was showed that the PCNA expressed by RGV is exclusively distributed with the viral nuclei at viral assembly sites. Therefore, the result suggests that RGV PCNA might be a viral protein interacting with viral nuclei.