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Global landscape of structural proteins of infectious spleen and kidney necrosis virus.

ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus in the family Iridoviridae, causes severe damage to mandarin fish cultures in China. Little is known about the proteins of ISKNV virions. In this study, a total of 38 ISKNV virion-associated proteins were identified by four different workflows with systematic and comprehensive proteomic approaches. Among the 38 identified proteins, 21 proteins were identified by the gel-based workflows (one-dimensional [1-D] and two-dimensional [2-D] gel electrophoresis). Fifteen proteins were identified by 1-D gel electrophoresis, and 16 proteins were identified by 2-D gel electrophoresis, with 10 proteins identified by both methods. Another 17 proteins were identified only by liquid chromatography (LC)-based workflows (LC-matrix-assisted laser desorption ionization [MALDI] and linear trap quadrupole [LTQ]-Orbitrap). Among these 17 LC-identified proteins, 5 proteins were identified uniquely by the LC-MALDI workflow, whereas another 6 proteins were identified only by the LTQ-Orbitrap workflow. These results underscore the importance of incorporation of multiple approaches in identification of viral proteins. Based on viral genomic sequence, genes encoding these 38 viral proteins were cloned and expressed in vitro. Antibodies were produced against these 38 proteins to confirm the ISKNV structural proteins by Western blotting. Of the newly identified proteins, ORF 056L and ORF 118L were identified and confirmed as two novel viral envelope proteins by Western blotting and immunoelectron microscopy (IEM). The ISKNV proteome reported here is currently the only characterized megalocytivirus proteome. The systematic and comprehensive identification of ISKNV structural proteins and their localizations in this study will facilitate future studies of the ISKNV assembly process and infection mechanism.

SUBMITTER: Dong CF

PROVIDER: S-EPMC3067969 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Global landscape of structural proteins of infectious spleen and kidney necrosis virus.

Dong Chuan-Fu CF Xiong Xiao-Peng XP Shuang Fan F Weng Shao-Ping SP Zhang Jing J Zhang Ye Y Luo Yong-Wen YW He Jian-Guo JG

Journal of virology 20110105 6

Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus in the family Iridoviridae, causes severe damage to mandarin fish cultures in China. Little is known about the proteins of ISKNV virions. In this study, a total of 38 ISKNV virion-associated proteins were identified by four different workflows with systematic and comprehensive proteomic approaches. Among the 38 identified proteins, 21 proteins were identified by the gel-based workflows (one-dimensi ...[more]

PMID: 21209107

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Project description:Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus Megalocytivirus, family Iridoviridae, brings great harm to fish farming. In infected tissues, ISKNV infection is characterized by a unique phenomenon, in that the infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to wall off the infected cells from host immune attack. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct a basement membrane (BM)-like structure, termed virus-mock basement membrane (VMBM), on the surface of infected cells to provide attaching sites for LECs. VMBMs do not contain collagen IV protein, which is essential for maintenance of BM integrity and functions. In this study, we identified the VP08R protein encoded by ISKNV. VP08R was predicted to be a secreted protein with a signal peptide but without a transmembrane domain. However, immunofluorescence assays demonstrated that VP08R is located on the plasma membrane of infected cells and shows an expression profile similar to that of VP23R. Coimmunoprecipitation showed that VP08R interacts with both VP23R and nidogen-1, indicating that VP08R is a component of VMBM and is present on the cell membrane by binding to VP23R. Through formation of intermolecular disulfide bonds, VP08R molecules self-organized into a multimer, which may play a role in the maintenance of VMBM integrity and stability. Moreover, the VP08R multimer was easily degraded when the ISKNV-infected cells were lysed, which may be a mechanism for VMBM disassembly when necessary to free LECs and release the mature virions.Infectious spleen and kidney necrosis virus (ISKNV; genus Megalocytivirus, family Iridovirus) is most harmful to cultured fishes. In tissues, the ISKNV-infected cells are attached by lymphatic endothelial cells (LECs), which are speculated to segregate the host immune system. A viral membrane protein, VP23R, binds and recruits the host nidogen-1 protein to construct virus-mock basement membranes (VMBMs) on the surface of infected cells to provide attaching sites for LECs. Although VMBMs lack the collagen IV network, which is an essential structural part of true BMs, VMBMs still show an intact structure. An ISKNV-encoded VP08R protein can self-assemble into a multimer and bind both VP23R and nidogen-1 to maintain the integrity and stability of VMBMs. On the basis of these facts, we redrew the putative schematic illustration of the VMBM structure. Our study suggests that the virus adopts a strategy to remodel the cellular matrix and may provide an important reference to elucidate BM functions and the mechanisms of lymphangiogenesis.

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Global landscape of structural proteins of infectious spleen and kidney necrosis virus.

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Global landscape of structural proteins of infectious spleen and kidney necrosis virus.

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