Cloning, expression and subcellular distribution of a Rana grylio virus late gene encoding ERV1 homologue.
ABSTRACT An essential for respiration and viability (ERV1) homologue, 88R, was cloned and characterized from Rana grylio virus (RGV). Database searches found its homologues in all sequenced iridoviruses, and sequence alignment revealed a highly conserved motif shared by all ERV1 family proteins: Cys-X-X-Cys. RT-PCR and western blot analysis revealed that 88R begins to transcribe and translate at 6 h postinfection (p.i.) and remains detectable at 48 h p.i. during RGV infection course. Furthermore, using drug inhibition analysis by a de novo protein synthesis inhibitor and a viral DNA replication inhibitor, RGV 88R was classified as a late (L) viral gene during the in vitro infection. 88R-EGFP fusion protein was observed in both the cytoplasm and nucleus of pEGFP-N3-88R transfected EPC cells. Although result of immunofluorescence is similar, 88R protein was not detected in viromatrix. Moreover, function of RGV 88R on virus replication were evaluated by RNAi assay. Nevertheless, effect of knockdown of RGV 88R expression on virus replication was not detected in cultured fish cell lines. Collectively, current data indicate that RGV 88R was a late gene of iridovirus encoding protein that distributed both the cytoplasm and nucleus.
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ABSTRACT: BACKGROUND: Replication and assembly of vertebrate reoviruses occur in specific intracellular compartments known as viral factories. Recently, NS88 and NS80, the nonstructural proteins from aquareoviruses, have been proposed to share common traits with µNS from orthoreoviruses, which are involved in the formation of viral factories. METHODOLOGYPRINCIPAL FINDINGS: In this study, the NS80 characteristics and its interactions with other viral components were investigated. We observed that the NS80 structure ensured its self-aggregation and selective recruitment of viral proteins to viral factories like structures (VFLS). The minimum amino acids (aa) of NS80 required for VFLS formation included 193 aa at the C-terminal. However, this truncated protein only contained one aa coil and located in the nucleus. Its N-terminal residual regions, aa 1-55 and aa 55-85, were required for recruiting viral nonstructural protein NS38 and structural protein VP3, respectively. A conserved N-terminal region of NS38, which was responsible for the interaction with NS80, was also identified. Moreover, the minimal region of C-terminal residues, aa 506-742 (Δ505), required for NS80 self-aggregation in the cytoplasm, and aa 550-742 (Δ549), which are sufficient for recruiting viral structure proteins VP1, VP2, and VP4 were also identified. CONCLUSIONSSIGNIFICANCE: The present study shows detailed interactions between NS80 and NS38 or other viral proteins. Sequence and structure characteristics of NS80 ensures its self-aggregation to form VFLS (either in the cytoplasm or nucleus) and recruitment of viral structural or nonstructural proteins.PLoS ONE 05/2013; 8(5):e63737. DOI:10.1371/journal.pone.0063737 · 3.53 Impact Factor
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ABSTRACT: Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, an envelope protein gene, 2L, was identified from Rana grylio virus (RGV). Its possible role in virus infection was investigated. Database searches found that RGV 2L had homologues in all sequenced iridoviruses and is a core gene of iridoviruses. Western blotting detection of purified RGV virions confirmed that 2L protein was associated with virion membrane. Fluorescence localization found that 2L protein co-localized with viral factories in RGV infected cells and presented as globular inclusions in transfected cells. In co-transfected cells, 2L protein co-localized with two other viral envelope proteins, 22R and 53R, respectively. However, 2L protein did not co-localize with the major capsid protein (MCP) of RGV in co-transfected cells. Meanwhile, fluorescence observation showed that 2L protein co-localized with endoplasmic reticulum, but did not co-localize with mitochondria and Golgi apparatus. Moreover, a conditional lethal mutant virus containing the lac repressor/operator system was constructed to investigate the role of RGV 2L in virus infection. The ability of plaques formation and the virus titers were strongly reduced when the expression of 2L was repressed. Therefore, the current data showed that 2L protein is essential for virus infection. Our study is the first report of co-localization between envelope proteins in iridovirus and would provide new insights into the understanding of envelope proteins in iridovirus.Journal of General Virology 12/2013; DOI:10.1099/vir.0.058776-0 · 3.53 Impact Factor
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ABSTRACT: Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, a recombinant RGV (i53R-RGV-lacIO) containing the inducible lac repressor/operator system was constructed. i53R-RGV-lacIO was a conditional lethal mutant in which the expression of envelope protein 53R was regulated by IPTG. i53R-RGV-lacIO shared characteristics similar to RGV in the presence of IPTG. However, the expression level of 53R, the ability of plaques formation, and the virus titers were strongly reduced in the absence of IPTG. Electron microscopy showed that the number of progeny virus produced by i53R-RGV-lacIO was remarkably reduced without IPTG. Furthermore, over-expression of 53R in vitro could increase titers of i53R-RGV-lacIO in the absence of IPTG. Therefore, the current data suggested that the lac repressor/operator system could regulate gene expression in the recombinant iridovirus. Our study was thought to be the first report of the system in aquatic virus.Virus Research 07/2013; 177(2). DOI:10.1016/j.virusres.2013.07.016 · 2.83 Impact Factor