Lymphocystis Disease Virus (Iridoviridae) Enters Flounder (Paralichthys olivaceus) Gill Cells via a Caveolae-Mediated Endocytosis Mechanism Facilitated by Viral Receptors

In previous research, voltage-dependent anion channel protein 2 (VDAC2) and the receptor of activated protein C kinase 1 (RACK1) in flounder (Paralichthys olivaceus) were confirmed as functional receptors for lymphocystis disease virus (LCDV) entry; however, the underlying mechanism of VDAC2- and RACK1-mediated LCDV entry remains unclear. In this study, we elucidated the endocytosis pathway of LCDV entry into flounder gill (FG) cells by treatment with specific inhibitory agents, siRNAs, and co-localization analysis. LCDV entry was significantly inhibited by the disruption of caveolae-mediated endocytosis, dynamin, and microtubules, and the knockdown of caveoline-1 and dynamin expression, but was not inhibited by the disruption of clathrin-mediated endocytosis, micropinocytosis, or low-pH conditions. The disruption of caveolae-mediated and clathrin-mediated endocytosis was verified by the internalization of cholera toxin subunit B (CTB) and transferrin, respectively. Confocal immunofluorescence assay demonstrated that LCDV was co-localized with VDAC2 and RACK1, CTB was co-localized with VDAC2 and RACK1 and partially with LCDV, but transferrin was not co-localized with LCDV, VDAC2, or RACK1, indicating that LCDV utilized the same pathway as CTB, i.e., caveolae-mediated endocytosis. This was different from the pathway of transferrin, which used clathrin-mediated endocytosis. Furthermore, caveolin-1 was co-localized with LCDV, VDAC2, and RACK1, suggesting that caveolin-1 was involved in LCDV entry. These results revealed for the first time that LCDV entered into FG cells via caveolae-mediated endocytosis facilitated by VDAC2 and RACK1 receptors, relying on dynamin and microtubules in a pH-independent manner, which provided new insight into the molecular mechanisms of LCDV entry and potential for the development of antiviral agents, expanding our understanding of iridovirus infection.

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Voltage-Dependent Anion Channel Protein 2 (VDAC2) and Receptor of Activated Protein C Kinase 1 (RACK1) Act as Functional Receptors for Lymphocystis Disease Virus Infection

Journal of Virology ◽

10.1128/jvi.00122-19 ◽

2019 ◽

Vol 93 (12) ◽

Cited By ~ 4

Author(s):

Ying Zhong ◽

Xiaoqian Tang ◽

Xiuzhen Sheng ◽

Jing Xing ◽

Wenbin Zhan

Keyword(s):

Disease Virus ◽

Protein C ◽

Polyclonal Antibodies ◽

Activated Protein C ◽

Anion Channel ◽

Voltage Dependent Anion Channel ◽

Lymphocystis Disease Virus ◽

Channel Protein ◽

Lymphocystis Disease ◽

Voltage Dependent

ABSTRACTIn previous research, a 27.8-kDa protein in flounderParalichthys olivaceusgill (FG) cells was identified as a putative cellular receptor (27.8R), which mediated lymphocystis disease virus (LCDV) infection via interaction with a 32-kDa viral attachment protein (VAP) of LCDV, and monoclonal antibodies (MAbs) against 27.8R and 32-kDa VAP were developed. In this study, the 27.8R was identified as voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1) of flounder. Recombinant VDAC2 (rVDAC2) and RACK1 (rRACK1) were obtained by prokaryotic expression, and rabbit anti-VDAC2/RACK1 polyclonal antibodies were prepared. The rVDAC2, rRACK1, and 27.8-kDa proteins in FG cells were recognized by anti-27.8R MAbs and anti-VDAC2/RACK1 polyclonal antibodies simultaneously. Preincubation of FG cells with anti-VDAC2/RACK1 polyclonal antibodies significantly decreased the percentages of LCDV-infected cells and LCDV copy numbers, blocked virus infection, and delayed the development of cytopathic effect. The mRNA expressions of VDAC2 and RACK1 in FG cells were upregulated to maximum levels 12 h and 48 h after LCDV infection, respectively. VDAC2/RACK1 knockdown through short interfering RNA (siRNA) significantly reduced VDAC2/RACK1 expression and LCDV copy numbers in FG cells compared with negative controls, while VDAC2/RACK1 expression on LCDV-nonpermissive epithelial papillosum cells (EPCs) conferred susceptibility to LCDV infection, indicating the VDAC2 and RACK1 were sufficient to allow LCDV entry and infection. All these results collectively showed that VDAC2 and RACK1 function as receptors for LCDV entry and infection.IMPORTANCELymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease in fish, which has caused huge economic losses to the aquaculture industry worldwide, but the molecular mechanism underlying the LCDV-host interaction remains unclear. Here, the 27.8-kDa putative cellular receptor for LCDV was identified as voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1), and our results revealed that VDAC2 and RACK1 expression was sufficient to allow LCDV entry and that they are functional receptors that initiate LCDV infection for the first time, which leads to a better understanding of the molecular mechanism underlying LCDV infection and virus-host interactions.

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Effect of probiotics enriched diet on Paralichthys olivaceus infected with lymphocystis disease virus (LCDV)

Fish & Shellfish Immunology ◽

10.1016/j.fsi.2010.07.031 ◽

2010 ◽

Vol 29 (5) ◽

pp. 868-874 ◽

Cited By ~ 79

Author(s):

Ramasamy Harikrishnan ◽

Chellam Balasundaram ◽

Moon-Soo Heo

Keyword(s):

Disease Virus ◽

Paralichthys Olivaceus ◽

Lymphocystis Disease Virus ◽

Lymphocystis Disease

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Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Chinese Journal of Oceanology and Limnology ◽

10.1007/s00343-011-0060-4 ◽

2011 ◽

Vol 29 (3) ◽

pp. 603-608

Author(s):

Mingqing Xing ◽

Xiuqin Sun ◽

Fengrong Zheng ◽

Lingyun Qu ◽

Xuguang Hong ◽

...

Keyword(s):

Disease Virus ◽

Paralichthys Olivaceus ◽

Japanese Flounder ◽

Lymphocystis Disease Virus ◽

Lymphocystis Disease

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Development and Characterization of Monoclonal Antibodies to the 32 kDa Viral Attachment Protein of Lymphocystis Disease Virus and Their Neutralizing Ability in Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms19092536 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2536 ◽

Cited By ~ 2

Author(s):

Ying Zhong ◽

Xiaoqian Tang ◽

Xiuzhen Sheng ◽

Jing Xing ◽

Wenbin Zhan

Keyword(s):

Monoclonal Antibodies ◽

Disease Virus ◽

Paralichthys Olivaceus ◽

Enzyme Linked Immunosorbent Assay ◽

Dilution Method ◽

Attachment Protein ◽

Lymphocystis Disease Virus ◽

Viral Attachment ◽

Lymphocystis Disease

In previous research, a 32 kDa protein in lymphocystis disease virus (LCDV) was identified as viral attachment protein (VAP) that specifically interacted with the 27.8 kDa cellular receptor from flounder Paralichthys olivaceus gill (FG) cells, and the recombinant VAP (rVAP) was expressed in Escherichia coli strain BL21 (DE3). In this study, monoclonal antibodies (MAbs) against 32 kDa VAP are produced by immunization of BALB/c mice with the rVAP. Seven hybridoma secreting MAbs were screened by enzyme-linked immunosorbent assay, five of which designated as 1C6, 1C8, 3B5, 3D11 and 3H10 are cloned by the limiting dilution method, depending on the strongly positive results of ELISA. Western blotting analysis shows that the five MAbs can specifically react with the 32 kDa protein of LCDV and the purified 50 kDa rVAP, and the subtype of the MAbs is identified as IgG. Immunofluorescence results demonstrate that the specific fluorescence signals for LCDV appear in the cytoplasm of FG cells at 24 h post LCDV infection. Neutralization assay results indicate that pre-incubations of LCDV with the five MAbs can significantly decrease the LCDV copy numbers and delay the development of the cytopathic effect in FG cells, revealing that the five MAbs can neutralize the LCDV particles and block viral infection in vitro. The neutralizing MAbs against 32 kDa VAP would be useful for the study on the LCDV–host interaction and might be promising inhibitors of LCDV infection in fish.

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