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The roles of grouper clathrin light chains in regulating the infection of a novel marine DNA virus, Singapore grouper iridovirus.

ABSTRACT: Clathrins, composed of clathrin heavy chains (CHCs) and clathrin light chains (CLCs), are usually hijacked by viruses for infection. However, the role of CLCs, especially in regulating fish virus infection, remains poorly understood. Here, two isoforms of CLCs were cloned from the red-spotted grouper (Epinephelus akaara) (EaCLCa and EaCLCb). Both EaCLC transcripts were expressed in all examined tissues, and the expression of EaCLCa was much higher than that of EaCLCb. Over-expressing EaCLCa-W119R mutant significantly reduced Singapore grouper iridovirus (SGIV) infectivity. However, no effect of EaCLCb-W122R on SGIV infection was observed. The detailed steps were further studied, mainly including virus attachment, entry and the following transport to early endosomes. EaCLCa-W119R mutant notably inhibited internalization of SGIV particles with no effect on SGIV attachment. Furthermore, EaCLCa-W119R mutant obviously impaired the delivery of SGIV to early endosomes after virus internalization. In addition, the EaCLCa-W119R mutant markedly reduced the colocalization of SGIV and actin. However, EaCLCb is not required for such events during SGIV infection. Taken together, these results demonstrate for the first time that EaCLCa and EaCLCb exerted different impacts on iridovirus infection, providing a better understanding of the mechanisms of SGIV infection and opportunities for the design of new antiviral strategies.

SUBMITTER: Wang L

PROVIDER: S-EPMC6821850 | biostudies-other | 2019 Oct

REPOSITORIES: biostudies-other

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The roles of grouper clathrin light chains in regulating the infection of a novel marine DNA virus, Singapore grouper iridovirus.

Wang Liqun L Li Qiang Q Ni Songwei S Huang Youhua Y Wei Jingguang J Liu Jiaxin J Yu Yepin Y Wang Shaowen S Qin Qiwei Q

Scientific reports 20191030 1

Clathrins, composed of clathrin heavy chains (CHCs) and clathrin light chains (CLCs), are usually hijacked by viruses for infection. However, the role of CLCs, especially in regulating fish virus infection, remains poorly understood. Here, two isoforms of CLCs were cloned from the red-spotted grouper (Epinephelus akaara) (EaCLCa and EaCLCb). Both EaCLC transcripts were expressed in all examined tissues, and the expression of EaCLCa was much higher than that of EaCLCb. Over-expressing EaCLCa-W119 ...[more]

PMID: 31666545

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Project description:BackgroundMicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms.Methodology/principal findingsTo determine whether SGIV encoded miRNAs during infection, a small RNA library derived from SGIV-infected grouper (GP) cells was constructed and sequenced by Illumina/Solexa deep-sequencing technology. We recovered 6,802,977 usable reads, of which 34,400 represented small RNA sequences encoded by SGIV. Sixteen novel SGIV-encoded miRNAs were identified by a computational pipeline, including a miRNA that shared a similar sequence to herpesvirus miRNA HSV2-miR-H4-5p, which suggests miRNAs are conserved in far related viruses. Generally, these 16 miRNAs are dispersed throughout the SGIV genome, whereas three are located within the ORF057L region. Some SGIV-encoded miRNAs showed marked sequence and length heterogeneity at their 3' and/or 5' end that could modulate their functions. Expression levels and potential biological activities of these viral miRNAs were examined by stem-loop quantitative RT-PCR and luciferase reporter assay, respectively, and 11 of these viral miRNAs were present and functional in SGIV-infected GP cells.ConclusionsOur study provided a genome-wide view of miRNA production for iridoviruses and identified 16 novel viral miRNAs. To the best of our knowledge, this is the first experimental demonstration of miRNAs encoded by aquatic animal viruses. The results provide a useful resource for further in-depth studies on SGIV infection and iridovirus pathogenesis.

Project description:UnlabelledIridoviruses 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-β-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.

Dataset Information

The roles of grouper clathrin light chains in regulating the infection of a novel marine DNA virus, Singapore grouper iridovirus.

Publications

The roles of grouper clathrin light chains in regulating the infection of a novel marine DNA virus, Singapore grouper iridovirus.

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