Article

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: 4.65). 02/2005; 79(1):140-9. DOI: 10.1128/JVI.79.1.140-149.2005
Source: PubMed

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.

Download full-text

Full-text

Available from: Jiann-Horng Leu, Feb 05, 2014
0 Followers
 · 
84 Views
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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 · 3.53 Impact Factor
  • Source
    • "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). "
    Journal of General Virology 01/2010; · 3.53 Impact Factor
  • Source
    • "It can cause 100% mortality within 3–10 days from the onset of visible gross signs [6] [7]. WSSV is an enveloped, large circular double-stranded DNA virus that expresses six major and about 34 minor proteins [8] [9] [10], of which VP19 and VP28 are the major WSSV envelope proteins involved in systemic virus infection [11]. A number of strategies for prophylaxis and control of WSSV have been explored but each with some limitations. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The use of stable and cost-effective DNA vaccines for silencing genes of White Spot Syndrome Virus (WSSV) in Penaeus monodon through RNA interference has been tested. DNA constructs that express long-hairpin RNA (lhRNA) constitutively in vivo against two WSSV envelop genes vp19 and vp28 (pCMV-VP19-LH and pCMV-VP28-LH) were used to confer protection against the pathogen. A construct expressing lhRNA directed against gfp gene (pCMV-GFP-LH) was used to estimate the non-specific protection and to show that on intramuscular administration the plasmid spreads all across the shrimp body and persists for at least 30 days. Shrimp injected with 10 microg lhRNA expressing constructs were challenged with purified WSSV after 24h and pCMV-VP28-LH provided best protection as evidenced by higher survival, lower viral load and silencing of the target viral gene. At an increased dose (35 microg), this construct showed better protection with 75% survival and 81% silencing of the target vp28 mRNA. The construct could actually reduce the viral copy number from the initial 10(3) to 10(5) copies to 10-100 copies. The non-specific pCMV-GFP-LH also showed improved survival and silencing of viral genes, although its effect was less than one third of pCMV-VP28-LH. Silencing constructs can be a good prophylactic approach in commercial shrimp hatcheries to protect high value brooders and to check the vertical transmission of virus, which is a major route of viral entry. Silencing by lhRNA expressed in vivo, confirms the presence of a functional microRNA pathway in shrimp.
    Vaccine 07/2009; 27(29):3849-55. DOI:10.1016/j.vaccine.2009.04.011 · 3.49 Impact Factor
Show more