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

Institute of Zoology, National Taiwan University, Taipei, Taiwan, Republic of China.
Journal of Virology (Impact Factor: 4.44). 02/2005; 79(1):140-9. DOI: 10.1128/JVI.79.1.140-149.2005
Source: PubMed


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.

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Available from: Jiann-Horng Leu, Feb 05, 2014
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    • "Also ORF62, a giant ORF was identified as encoding a constituent of GpSGHV virions representing a protein of about 511 kDa. Such a large virion protein is not unusual for large dsDNA viruses, as white spot syndrome virus has a 664 kDa virion protein that is a major nucleocapsid protein (Leu et al., 2005; van Hulten et al., 2001). Whereas the 511 kDa protein is probably a minor component (1.8 % peptide coverage), another large protein of 127 kDa (ORF10) was found in high abundance (with 16.9 % coverage) and probably represents the 130 kDa protein seen in SDS-PAGE (Fig. 2a). "
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    ABSTRACT: Many species of tsetse flies (Diptera: Glossinidae) can be infected by a virus that causes salivary gland hypertrophy (SGH). The genomes of viruses isolated from Glossina pallidipes (GpSGHV) and Musca domestica (MdSGHV) have recently been sequenced. Tsetse flies with SGH have reduced fecundity and fertility which cause a serious problem for mass rearing in the frame of sterile insect technique (SIT) programmes to control and eradicate tsetse populations in the wild. A potential intervention strategy to mitigate viral infections in fly colonies is neutralizing of the GpSGHV infection with specific antibodies against virion proteins. Two major GpSGHV virion proteins of about 130 and 50 kDa, respectively, were identified by Western analysis using a polyclonal rabbit antibody raised against whole GpSHGV virions. The proteome of GpSGHV, containing the antigens responsible for the immune-response, was investigated by liquid chromatography tandem mass spectrometry and 61 virion proteins were identified by comparison with the genome sequence. Specific antibodies were produced in rabbits against seven candidate proteins, including the ORF10/C-terminal fragment, ORF47 and ORF96 as well as proteins involved in peroral infectivity PIF-1 (ORF102), PIF-2 (ORF53), PIF-3 (ORF76) and P74 (ORF1). Antiserum against ORF10 specifically reacted to the 130 kDa protein in a Western blot analysis and to the envelope protein of GpSGHV, detected by using immunogold-electron microscopy. This result suggests that immune intervention of viral infections in colonies of G. pallidipes is a realistic option.
    Journal of General Virology 12/2010; 91(Pt 12):3065-74. DOI:10.1099/vir.0.023671-0 · 3.18 Impact Factor
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    • "However, the nucleocapsid's ultrastructure has not yet been clearly resolved. The stacked rings are formed by the nucleocapsid protein VP664 [38], but until now it has not been clear exactly how the protein molecules were related to the regular arrangement of globular shapes that made up each ring. Based on the observed mass (644 kDa) of VP664, the size-mass relationship found by Erickson [41] predicts a protein diameter of about 11.5 nm with a minimal radius (Rmin) of 5.76 nm. "
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    ABSTRACT: Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus), is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process. In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument) proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers. From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented.
    PLoS ONE 05/2010; 5(5):e10718. DOI:10.1371/journal.pone.0010718 · 3.23 Impact Factor
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    • "The causative agent of WSD is a unique, novel, large (approximately 300 kbp) dsDNA virus, white spot syndrome virus (WSSV) [2]. WSSV encodes several remarkable proteins, such as VP664 [3] [4], which is the largest viral structural protein ever found, and ICP11, which is the most highly expressed viral protein and also the rst viral DNA mimic protein to be reported [5] [6]. To date, however, there is still only limited data on how WSSV interacts with its hosts, and the pathogenesis of the disease is not completely understood. "
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