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

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.White spot syndrome virus (WSSV) is one of the most devastating shrimp pathogens, and it has caused serious damage to the worldwide shrimp culture industry. Although this virus can infect several crustacean species, including shrimp, crab, and crayfish (9,12,19,24,42), its virulence is particularly high in infected penaeid shrimp, and mortality can reach 90 to 100% within 3 to 7 days of infection (9, 46). The WSSV virion is a nonoccluded, enveloped particle of approximately 275 by 120 nm with an olive-to-bacilliform shape, and it has a nucleocapsid (300 by 70 nm) with periodic striations perpendicular to the long axis (42,43). The most prominent feature of WSSV is the presence of a tail-like extension at one end of the virion (10, 43), which gives this virus the family name Nimaviridae (27) ("nima" is Latin for "thread").The complete genome sequences of three WSSV isolates have been determined, and 532 open reading frames (ORFs) that contain Ͼ60 amino acids have been identified for the Taiwan isolate (GenBank accession number AF440570). Homology searches against the NCBInr database suggest possible roles or functions for only a few of these ORFs, and most of the ORFs posted for the Taiwan isolate show no significant similarity to other known proteins. Similar results have been reported for the other two isolates (40,44). Until recently, Ͻ5% of the ORFs had been shown to encode either nonstructural proteins (ribonucleotide reductase, protein kinase, the chimeric thymidine kinase/thymidylate kinase, and DNA polymerase [8,22,35,36]) or structural proteins...

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

“…Immunogold electron microscopy (IEM) has been used to identify VP28, VP26, VP31, VP51C, VP36B, VP41A, VP12B, and VP180 as envelope proteins (4,5,8,9,(28)(29)(30) and VP664 as a nucleocapsid protein (7). Other studies (1,6,(18)(19)(20) that combined detergent treatment and Western blotting confirmed and expanded most of these results (VP28, VP19, and VP73 as envelope proteins; VP24, VP15, and VP35 as nucleocapsid proteins) but also identified VP26 as a nucleocapsid protein.…”

“…To identify the proteins in the SDS-PAGE gels, polyclonal antibodies against specific WSSV structural proteins were generated either in rat (for VP26) or in rabbit (for 14 other proteins) by using the expressed recombinant proteins in Escherichia coli as described previously (7). The polyvinylidene difluoride membranes were then incubated with diluted primary antibody for 1 h at room temperature, and anti-rabbit or anti-rat immunoglobulin G antibody conjugated to horseradish peroxidase was used as the secondary antibody.…”

White Spot Syndrome Virus Annexes a Shrimp STAT To Enhance Expression of the Immediate-Early Gene ie1

“…WSSV is a large DNA virus with a virion that consists of a nucleocapsid, tegument, and envelope and includes at least 39 structural proteins (22,38,39,43). WSSV is extremely virulent (17,28,29), has a wide host range (12,26), and targets various tissues (25,26,42).…”

Protection against white spot syndrome virus (WSSV) infection in kuruma shrimp orally vaccinated with WSSV rVP26 and rVP28

“…The viral nucleocapsid contains a DNA-protein core bounded by a distinctive capsid layer and a single molecule of circular doublestranded DNA with an approximate size of 300 kbp (van Hulten et al 2001, Yang et al 2001. WSSV contains at least 6 major proteins: VP28 and VP19, which are associated with the envelope; VP664 and VP15, associated with the nucleocapsid; and VP24 and VP26, which are located in between the envelope and the nucleocapsid (van Hulten et al 2000a,b, Chen et al 2002, Leu et al 2005, Tsai et al 2006.…”