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Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly.

ABSTRACT: Singapore Grouper Iridovirus (SGIV) is a member of nucleo cytoplasmic large DNA viruses (NCLDV). This paper reports the functional analysis of ORF75R, a major structural protein of SGIV. Immuno fluorescence studies showed that the protein was accumulated in the viral assembly site. Immunogold-labelling indicated that it was localized between the viral capsid shell and DNA core. Knockdown of ORF75R by morpholinos resulted in the reduction of coreshell thickness, the failure of DNA encapsidation, and the low yield of infectious particles. Comparative proteomics further identified the structural proteins affected by ORF75R knockdown. Two-dimensional gel electrophoresis combined with proteomics demonstrated that ORF75R was phosphorylated at multiple sites in SGIV-infected cell lysate and virions, but the vast majority of ORF75R in virions was the dephosphorylated isoform. A kinase assay showed that ORF75R could be phosphorylated in vitro by the SGIV structural protein ORF39L. Addition of ATP and Mg(2+) into purified virions prompted extensive phosphorylation of structural proteins and release of ORF75R from virions. These data suggest that ORF75R is a novel scaffold protein important for viral assembly and DNA encapsidation, but its phosphorylation facilitates virion disassembly. Compared to proteins from other viruses, we found that ORF75R shares common features with herpes simplex virus VP22.

SUBMITTER: Wang F

PROVIDER: S-EPMC4541339 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly.

Wang Fan F Liu Yang Y Zhu Yi Y Ngoc Tran Bich B Wu Jinlu J Leong Hew Choy C

Scientific reports 20150819

Singapore Grouper Iridovirus (SGIV) is a member of nucleo cytoplasmic large DNA viruses (NCLDV). This paper reports the functional analysis of ORF75R, a major structural protein of SGIV. Immuno fluorescence studies showed that the protein was accumulated in the viral assembly site. Immunogold-labelling indicated that it was localized between the viral capsid shell and DNA core. Knockdown of ORF75R by morpholinos resulted in the reduction of coreshell thickness, the failure of DNA encapsidation, ...[more]

PMID: 26286371

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Project description:BackgroundOrange-spotted grouper (Epinephelus coioides) is an economically important marine fish cultured in China and Southeast Asian countries. The emergence of infectious viral diseases, including iridovirus and betanodavirus, have severely affected food products based on this species, causing heavy economic losses. Limited available information on the genomics of E. coioides has hampered the understanding of the molecular mechanisms that underlie host-virus interactions. In this study, we used a 454 pyrosequencing method to investigate differentially-expressed genes in the spleen of the E. coioides infected with Singapore grouper iridovirus (SGIV).ResultsUsing 454 pyrosequencing, we obtained abundant high-quality ESTs from two spleen-complementary DNA libraries which were constructed from SGIV-infected (V) and PBS-injected fish (used as a control: C). A total of 407,027 and 421,141 ESTs were produced in control and SGIV infected libraries, respectively. Among the assembled ESTs, 9,616 (C) and 10,426 (V) ESTs were successfully matched against known genes in the NCBI non-redundant (nr) database with a cut-off E-value above 10-5. Gene ontology (GO) analysis indicated that "cell part", "cellular process" and "binding" represented the largest category. Among the 25 clusters of orthologous group (COG) categories, the cluster for "translation, ribosomal structure and biogenesis" represented the largest group in the control (185 ESTs) and infected (172 ESTs) libraries. Further KEGG analysis revealed that pathways, including cellular metabolism and intracellular immune signaling, existed in the control and infected libraries. Comparative expression analysis indicated that certain genes associated with mitogen-activated protein kinase (MAPK), chemokine, toll-like receptor and RIG-I signaling pathway were alternated in response to SGIV infection. Moreover, changes in the pattern of gene expression were validated by qRT-PCR, including cytokines, cytokine receptors, and transcription factors, apoptosis-associated genes, and interferon related genes.ConclusionThis study provided abundant ESTs that could contribute greatly to disclosing novel genes in marine fish. Furthermore, the alterations of predicted gene expression patterns reflected possible responses of these fish to the virus infection. Taken together, our data not only provided new information for identification of novel genes from marine vertebrates, but also shed new light on the understanding of defense mechanisms of marine fish to viral pathogens.

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Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly.

Publications

Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly.

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