scholarly journals Identification of the Nucleocapsid, Tegument, and Envelope Proteins of the Shrimp White Spot Syndrome Virus Virion

2006 ◽  
Vol 80 (6) ◽  
pp. 3021-3029 ◽  
Author(s):  
Jyh-Ming Tsai ◽  
Han-Ching Wang ◽  
Jiann-Horng Leu ◽  
Andrew H.-J. Wang ◽  
Ying Zhuang ◽  
...  
Keyword(s):  
White Spot Syndrome Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Envelope Proteins ◽  
White Spot ◽  
Structural Proteins ◽  
Nucleocapsid Proteins ◽  
Tegument Proteins ◽  
White Spot Syndrome ◽  
Protein Components

ABSTRACT The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.

scholarly journals Proteomic Analysis of the Major Envelope and Nucleocapsid Proteins of White Spot Syndrome Virus

2006 ◽  
Vol 80 (21) ◽  
pp. 10615-10623 ◽  
Author(s):  
Xixian Xie ◽  
Limei Xu ◽  
Feng Yang
Keyword(s):  
Posttranslational Modifications ◽  
White Spot Syndrome Virus ◽  
Procambarus Clarkii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
White Spot ◽  
Structural Proteins ◽  
Nucleocapsid Proteins ◽  
Flight Mass Spectrometry ◽  
White Spot Syndrome

ABSTRACT White spot syndrome virus (WSSV) virions were purified from the tissues of infected Procambarus clarkii (crayfish) isolates. Pure WSSV preparations were subjected to Triton X-100 treatment to separate into the envelope and nucleocapsid fractions, which were subsequently separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major envelope and nucleocapsid proteins were identified by either matrix-assisted laser desorption ionization-time of flight mass spectrometry or defined antibody. A total of 30 structural proteins of WSSV were identified in this study; 22 of these were detected in the envelope fraction, 7 in the nucleocapsid fraction, and 1 in both the envelope and the nucleocapsid fractions. With the aid of specific antibodies, the localizations of eight proteins were further studied. The analysis of posttranslational modifications revealed that none of the WSSV structural proteins was glycosylated and that VP28 and VP19 were threonine phosphorylated. In addition, far-Western and coimmunoprecipitation experiments showed that VP28 interacted with both VP26 and VP24. In summary, the data obtained in this study should provide an important reference for future molecular studies of WSSV morphogenesis.

scholarly journals Crystal Structures of Major Envelope Proteins VP26 and VP28 from White Spot Syndrome Virus Shed Light on Their Evolutionary Relationship

2007 ◽  
Vol 81 (12) ◽  
pp. 6709-6717 ◽  
Author(s):  
Xuhua Tang ◽  
Jinlu Wu ◽  
J. Sivaraman ◽  
Choy Leong Hew
Keyword(s):  
Viral Infection ◽  
White Spot Syndrome Virus ◽  
Evolutionary Relationship ◽  
Viral Proteins ◽  
Envelope Proteins ◽  
Viral Envelope ◽  
White Spot ◽  
Structural Proteins ◽  
White Spot Syndrome ◽  
Effective Transfer

ABSTRACT White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 Å, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt β-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core β-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.

scholarly journals Genomic and Proteomic Analysis of Thirty-Nine Structural Proteins of Shrimp White Spot Syndrome Virus

2004 ◽  
Vol 78 (20) ◽  
pp. 11360-11370 ◽  
Author(s):  
Jyh-Ming Tsai ◽  
Han-Ching Wang ◽  
Jiann-Horng Leu ◽  
He-Hsuan Hsiao ◽  
Andrew H.-J. Wang ◽  
...  
Keyword(s):  
White Spot Syndrome Virus ◽  
Expression Patterns ◽  
Sodium Dodecyl ◽  
Peptide Sequence ◽  
White Spot ◽  
Structural Proteins ◽  
Structural Protein ◽  
Structural Genes ◽  
Sds Page ◽  
White Spot Syndrome

ABSTRACT White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands were then excised from the gel, and following trypsin digestion of the reduced and alkylated WSSV proteins in the bands, the peptide sequence of each fragment was determined by liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) using a quadrupole/time-of-flight mass spectrometer. Comparison of the resulting peptide sequence data against the nonredundant database at the National Center for Biotechnology Information identified 33 WSSV structural genes, 20 of which are reported here for the first time. Since there were six other known WSSV structural proteins that could not be identified from the SDS-PAGE bands, there must therefore be a total of at least 39 (33 + 6) WSSV structural protein genes. Only 61.5% of the WSSV structural genes have a polyadenylation signal, and preliminary analysis by 3′ rapid amplification of cDNA ends suggested that some structural protein genes produced mRNA without a poly(A) tail. Microarray analysis showed that gene expression started at 2, 6, 8, 12, 18, 24, and 36 hpi for 7, 1, 4, 12, 9, 5, and 1 of the genes, respectively. Based on similarities in their time course expression patterns, a clustering algorithm was used to group the WSSV structural genes into four clusters. Genes that putatively had common or similar roles in the viral infection cycle tended to appear in the same cluster.

scholarly journals The Unique Stacked Rings in the Nucleocapsid of the White Spot Syndrome Virus Virion Are Formed by the Major Structural Protein VP664, the Largest Viral Structural Protein Ever Found

2005 ◽  
Vol 79 (1) ◽  
pp. 140-149 ◽  
Author(s):  
Jiann-Horng Leu ◽  
Jyh-Ming Tsai ◽  
Han-Ching Wang ◽  
Andrew H.-J. Wang ◽  
Chung-Hsiung Wang ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Time Course ◽  
White Spot Syndrome Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
White Spot ◽  
Sequencing Analysis ◽  
Structural Protein ◽  
Reading Frame ◽  
White Spot Syndrome

ABSTRACT One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.

Protein expression profiling in the gill of Litopenaeus vannamei (Boone, 1931) naturally infected with white spot syndrome virus

Crustaceana ◽  
2015 ◽  
Vol 88 (7-8) ◽  
pp. 747-765
Author(s):  
P. A. Valentim-Neto ◽  
A. P. M. Fraga ◽  
G. A. S. Müller ◽  
M. R. F. Marques
Keyword(s):  
Protein Expression ◽  
Litopenaeus Vannamei ◽  
White Spot Syndrome Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Differentially Expressed ◽  
White Spot ◽  
Mortality Factor ◽  
Control Group ◽  
Peptide Mass ◽  
White Spot Syndrome

To better understand the molecular pathogenesis of white spot syndrome virus (WSSV) inLitopenaeus vannamei(Boone, 1931), the protein expression profile in gills was characterized. Farmed shrimp WSSV positive were randomly sorted based on nested PCR. The proteomic analysis of gill proteins was performed using two-dimensional electrophoresis (2-DE), with isofocalisation on an immobilized linear gradient (pH 3-10), followed by separation based on molecular weight using 12.5% denaturating polyacrylamide gel electrophoresis (SDS-PAGE). The comparative analysis of the 2-DE profile between the two groups revealed eight differentially expressed spots in gills of naturally infected shrimp. The spots were successfully identified using MALDI-TOF mass spectrometry peptide mass fingerprint. The up-regulated proteins unique to infected shrimp were identified as peptidyl-prolyl isomerase, mortality factor 4-like protein 1, calreticulin, recombination activating protein, failed axon connection protein, 40S ribosomal S2 and N-deacetylase/N-sulfotransferase. The down-regulated protein unique to non-infected shrimp (control group) was identified as an inhibitor of apoptosis. The differentially expressed proteins are involved in several important cellular processes, such as host defence and protein metabolism. The present work contributes to a better understanding of the overall molecular responses elicited by WSSV infection inL. vannamei, as well as to point out potential molecular biomarkers to evaluate the susceptibility to the virus and the sanitary status in farmed shrimp.

scholarly journals Envelope Proteins of White Spot Syndrome Virus (WSSV) Interact with Litopenaeus vannamei Peritrophin-Like Protein (LvPT)

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0144922 ◽  
Author(s):  
Shijun Xie ◽  
Xiaojun Zhang ◽  
Jiquan Zhang ◽  
Fuhua Li ◽  
Jianhai Xiang
Keyword(s):  
Litopenaeus Vannamei ◽  
White Spot Syndrome Virus ◽  
Envelope Proteins ◽  
White Spot ◽  
White Spot Syndrome

scholarly journals Penaeidins are a novel family of antiviral effectors against WSSV in shrimp

2018 ◽  
Author(s):  
Bang Xiao ◽  
Qihui Fu ◽  
Shengwen Niu ◽  
Haoyang Li ◽  
Kai Lǚ ◽  
...  
Keyword(s):  
Viral Infection ◽  
Signaling Pathways ◽  
Outer Surface ◽  
Phagocytic Activity ◽  
White Spot Syndrome Virus ◽  
Antiviral Immunity ◽  
White Spot ◽  
Structural Proteins ◽  
First Report ◽  
White Spot Syndrome

AbstractPenaeidins are members of a family of key effectors with broad anti-bacterial activities in penaeid shrimp. However, the function of penaeidins in antiviral immunity is rarely reported and remains largely unknown. Herein, we uncovered that penaeidins are a novel family of antiviral effectors against white spot syndrome virus (WSSV). Firstly, RNAi in vivo mediated knockdown of each penaeidin from four identified penaeidins from Litopenaeus vannamei resulted in elevated viral loads and rendered shrimp more susceptible to WSSV, whilst the phenotype of survival rate in penaeidin-silenced shrimp can be rescued via the injection of recombinant penaeidin proteins. Moreover, pull-down assays demonstrated the conserved PEN domain of penaeidin was able to interact with WSSV structural proteins. Furthermore, we observed that colloidal gold-labeled penaeidins were located on the outer surface of the WSSV virion. By infection-blocking assay, we observed that hemocytes had lower viral infection rates in the group of WSSV preincubated with penaeidins than those of control group. Phagocytic activity analysis further showed that penaeidins were able to inhibit phagocytic activity of hemocytes against WSSV Taken together, these results suggest that penaeidins specifically binds to WSSV virion by interacting with its structural proteins, thus preventing viral infection that confers host against WSSV. In addition, dual-luciferase assay and EMSA assay demonstrated that penaeidins were regulated by Dorsal and Relish, two transcription factors of the canonical Toll and IMD pathway, respectively. To our best knowledge, this is the first report on uncovering the antiviral function of penaeidins in the innate immune system of shrimp.ImportancesWhite spot syndrome, caused by white spot syndrome virus (WSSV), is the most serious disease in shrimp aquaculture, which has long been a scourge of cultured shrimp industry. Herein, we provided some substantial evidences to indicate that penaeidins are a novel family of effectors with antiviral activity against WSSV in shrimp. Penaeidins such as BigPEN, PEN2 and PEN3 were able to interact with the outer surface of WSSV virion via binding to viral structural proteins, and thus preventing viral entry host cells. In addition, we demonstrated that the Toll and IMD signaling pathways can regulate the transcriptional expression of penaeidins, which may suggest an important role of the conserved innate signaling pathways in antiviral immunity. This is the first report of the antiviral mechanism of penaeidins in shrimp, which may provide some new insights into strategies to control WSSV infection in shrimp farms.

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