Characterization of an envelope gene VP19 from Singapore grouper iridovirus

Huang, Xiaohong; Gong, Jie; Huang, Youhua; Ouyang, Zhangxian; Wang, Shaowen; Chen, Xiuli; Qin, Qiwei

doi:10.1186/1743-422x-10-354

Cited by 36 publications

(18 citation statements)

References 40 publications

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“…3A). We pre- viously demonstrated that SGIV VP19 was an envelope protein that could be specifically recognized by antibody against VP19 during SGIV infection (58). In the present study, we also assessed the percentage of SGIV-infected cells under treatment with different drugs using IFA.…”

Section: Dynamics Of Sgiv Entry Into Gs Cells Virus Infection and Entrymentioning

confidence: 73%

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Entry of a Novel Marine DNA Virus, Singapore Grouper Iridovirus, into Host Cells Occurs via Clathrin-Mediated Endocytosis and Macropinocytosis in a pH-Dependent Manner

Wang

Huang

et al. 2014

J Virol

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103

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Iridoviruses are nucleocytoplasmic DNA viruses which cause great economic losses in the aquaculture industry but also show significant threat to global biodiversity. However, a lack of host cells has resulted in poor progress in clarifying iridovirus behavior. We investigated the crucial events during virus entry using a combination of single-virus tracking and biochemical assays, based on the established virus-cell infection model for Singapore grouper iridovirus (SGIV). SGIV infection in host cells was strongly inhibited when cells were pretreated with drugs blocking clathrin-mediated endocytosis, including sucrose and chlorpromazine. Inhibition of key regulators of macropinocytosis, including Na ؉ /H ؉ exchanger, Rac1 GTPase, p21-activated kinase 1 (PAK1), protein kinase C (PKC), and myosin II, significantly reduced SGIV uptake. Cy5-labeled SGIV particles were observed to colocalize with clathrin and macropinosomes. In contrast, disruption of cellular cholesterol by methyl-␤-cyclodextrin and nystatin had no effect on virus infection, suggesting that SGIV entered grouper cells via the clathrin-mediated endocytic pathway and macropinocytosis but not via caveola-dependent endocytosis. Furthermore, inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked virus infection, indicating that SGIV entered cells in a pH-dependent manner. In addition, SGIV particles were observed to be transported along both microtubules and actin filaments, and intracellular SGIV motility was remarkably impaired by depolymerization of microtubules or actin filaments. The results of this study for the first time demonstrate that not only the clathrin-dependent pathway but also macropinocytosis are involved in fish DNA enveloped virus entry, thus providing a convenient tactic for exploring the life cycle of DNA viruses. IMPORTANCEVirus entry into host cells is critically important for initiating infections and is usually recognized as an ideal target for the design of antiviral strategies. Iridoviruses are large DNA viruses which cause serious threats to ecological diversity and the aquaculture industry worldwide. However, the current understanding of iridovirus entry is limited and controversial. Singapore grouper iridovirus (SGIV) is a novel marine fish DNA virus which belongs to genus Ranavirus, family Iridoviridae. Here, using single-virus tracking technology in combination with biochemical assays, we investigated the crucial events during SGIV entry and demonstrated that SGIV entered grouper cells via the clathrin-mediated endocytic pathway in a pH-dependent manner but not via caveola-dependent endocytosis. Furthermore, we propose for the first time that macropinocytosis is involved in iridovirus entry. Together, this work not only contributes greatly to understating iridovirus pathogenesis but also provides an ideal model for exploring the behavior of DNA viruses in living cells.

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Section: Dynamics Of Sgiv Entry Into Gs Cells Virus Infection and Entrymentioning

confidence: 73%

“…Citrate buffer (40 mM sodium citrate, 10 mM KCl, and 135 mM NaCl, pH 3.1) was added and left for 45 s to inactivate and remove noninternalized viruses. Cells were examined at 48 h postinfection (hpi) by IFA using anti-VP19 antibody (58).…”

Section: Methodsmentioning

confidence: 99%

Entry of a Novel Marine DNA Virus, Singapore Grouper Iridovirus, into Host Cells Occurs via Clathrin-Mediated Endocytosis and Macropinocytosis in a pH-Dependent Manner

Wang

Huang

et al. 2014

J Virol

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103

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“…Moreover, the ectopic expressed Ec-c-Jun was translocated from nucleus to virus assembly sites. Given that virus assembly sites formed in iridovirus infection always contained numerous viral structural proteins, including membrane proteins and capsid proteins [35], we proposed that Ec-c-Jun might participate in SGIV infection via interaction with these viral proteins. During JCV infection, the transcription factors c-Jun physically and functionally interacted with JCV T-Ag and their interaction modulated JCV transcription and replication in glial cells [10].…”

Section: Discussionmentioning

confidence: 99%

Characterization of c-Jun from orange-spotted grouper, Epinephelus coioides involved in SGIV infection

Wei

Huang

et al. 2015

Fish & Shellfish Immunology

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The nuclear phosphoprotein c-Jun is a member of the AP1 family of transcription activating complex, can be induced by various extracellular stimuli such as virus infection. In this study, the c-Jun gene (Ec-c-Jun) was cloned from orange-spotted grouper, Epinephelus coioides. The full-length Ec-c-Jun cDNA is composed of 2046 bp and encodes a polypeptide of 328 amino acids with 81% identity of zebrafish. Amino acid alignment analysis indicated that Ec-c-Jun contained three conserved domains including a transactivation domain (TAD), a DNA-binding domain (DBD) and leucine zipper domain (LZD). RT-PCR results showed that Ec-c-Jun transcript was most abundant in spleen, kidney, heart and gill. The expression of Ec-c-Jun was up-regulated after challenged with Singapore grouper iridovirus (SGIV). To investigate the roles of Ec-c-Jun during SGIV infection, we constructed its dominant-negative mutant (DN-Ec-c-Jun) by deleting the major TAD that lacks amino acids 3-122. Fluorescence microscopy observation revealed that Ec-c-Jun and DN-Ec-c-Jun were expressed predominantly in the nucleus in transfected cells. Interestingly, the green fluorescence of Ec-c-Jun was congregated and co-localized with virus assembly sites at the late stage of SGIV infection. However, in DN-Ec-c-Jun transfected cells, no virus assembly sites were observed, and the distribution of fluorescence remained unchanged. Moreover, overexpression of DN-Ec-c-Jun in vitro delayed the occurrence of CPE induced by SGIV infection and inhibited the virus gene transcription. In addition, ectopic expression of DN-Ec-c-Jun was able to inhibit SGIV induced c-Jun/AP1 promoter activity in GS cells. Thus, we proposed that c-Jun transcription factor was essential for SGIV replication in vitro. Our results will contribute to understanding the crucial roles of JNK signaling pathway in fish virus infection.

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“…Two late genes, SGIV ORF088 and ORF019, encode viral envelope proteins. Furthermore, rVP88 was shown to bind a 94 kDa host cell membrane protein, suggesting that VP88 might function as an attachment protein and play a role in viral entry (Huang et al 2013a ;Zhou et al 2011 ). Finally, similar approaches have been applied to ISKNV (genus Megalocytivirus ) and used to identify putative functions among a viral TRAF protein (He et al 2012a ), a viral protein that mediates formation of a mock basement membrane and provides attachment sites for lymphatic endothelial cells (Xu et al 2010 ), a viral ankyrin repeat protein that may inhibit TNFα-induced NF-κB signaling (Guo et al 2011a ), and a viral-encoded vascular endothelial growth factor (Wang et al 2008c ).…”

Section: Erv1mentioning

confidence: 99%