Co-interactive DNA-binding between a novel, immunophilin-like shrimp protein and VP15 nucleocapsid protein of white spot syndrome virus

White spot syndrome virus (WSSV) is one of the most serious pathogens of penaeid shrimp. Although its genome has been completely characterized, the functions of most of its putative proteins are not yet known. It has been suggested that the major nucleocapsid protein VP15 is involved in packaging of the WSSV genome during virion formation. However, little is known in its relationship with shrimp host cells. Using the yeast two-hybrid approach to screen a shrimp lymphoid organ (LO) cDNA library for proteins that might interact with VP15, a protein named PmFKBP46 was identified. It had high sequence similarity to a 46 kDa-immunophilin called FKBP46 from the lepidopteran Spodoptera frugiperda (the fall armyworm). The full length PmFKBP46 consisted of a 1,257-nucleotide open reading frame with a deduced amino acid sequence of 418 residues containing a putative FKBP-PPIase domain in the C-terminal region. Results from a GST pull-down assay and histological co-localization revealed that VP15 physically interacted with PmFKBP46 and that both proteins shared the same subcellular location in the nucleus. An electrophoretic mobility shift assay indicated that PmFKBP46 possessed DNA-binding activity and functionally co-interacted with VP15 in DNA binding. The overall results suggested that host PmFKBP46 might be involved in genome packaging by viral VP15 during virion assembly.     

White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP)

White spot syndrome virus (WSSV) is the causative agent of a severe disease of cultivated shrimp. Using purified WSSV virions, VP53A encoded by open reading frame wssv067 was identified as a structural protein by SDS-PAGE and proteomics. Immunoelectron microscopy with a gold-labeled secondary antibody revealed that VP53A was distributed on the viral envelope. In order to further explore the link between WSSV067 and host proteins, we performed a yeast 2-hybrid screening of a Penaeus monodon cDNA library, using WSSV067C as bait. One of the molecules that specifically interacted with WSSV067C was the P. monodon chitin-binding protein (PmCBP). An in vitro binding assay showed that c-myc-WSSV067C was capable of co-precipitating HA-PmCBP-C. Furthermore, PmCBP was expressed in almost all organs but appeared to be up-regulated at the late stage of WSSV infection.     

WSSV: VP26 binding protein and its biological activity

White spot syndrome virus (WSSV) is one of the major causes of disease in the shrimp culture industry causing enormous economic losses. In this study, we displayed peptides from a cDNA library obtained from the hemolymph of shrimp infected with WSSV, on the surface of phage and screened for the peptides that interacted with the WSSV. One WSSV binding protein (WBP) gene was found to consist of 171 bp that had no matches in the NCBI database. This WBP was shown to bind to the VP26 protein of the WSSV by Western blotting. In addition, WBP reduced the binding of WSSV to shrimp haemocytes from 2.0 × 10(7)copies in the control to 6.0 × 10(2) after treatment with 80 μg of WBP. The survival rate of shrimp after WSSV were mixed with WBP at 80 μg, was 89% and the binding of WBP remained unchanged for at least 24h. Therefore, the results indicate that the WBP can bind to VP26 and inhibit the invasion of WSSV into host cells. This finding may introduce another future way to try to fight this disease in shrimp culture.     

Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466

White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV.     

Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus

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.     

Conservation of the DNA sequences encoding the major structural viral proteins of WSSV

A cDNA library was constructed from white spot syndrome virus (WSSV)-infected penaeid shrimp tissue. cDNA clones with WSSV inserts were isolated and sequenced. By comparison with DNA sequences in GenBank, cDNA clones containing sequence identical to those of the WSSV envelope protein VP28 and nucleoprotein VP15 were identified. Poly(A) sites in the mRNAs of VP28 and VP15 were identified. Genes encoding the major viral structural proteins VP28, VP26, VP24, VP19 and VP15 of 5 WSSV isolates collected from different shrimp species and/or geographical areas were sequenced and compared with those of 4 other WSSV isolate sequences in GenBank. For each of the viral structural protein genes compared, the nucleotide sequences were 100 to 99% identical among the 9 isolates. Gene probes or PCR primers based on the gene sequences of the WSSV structural proteins can be used for diagnoses and/or detection of WSSV infection.     

Transcriptional regulation of extracellular copper zinc superoxide dismutase from white shrimp Litopenaeus vannamei following Vibrio alginolyticus and WSSV infection

The cDNA encoding an extracellular copper zinc superoxide dismutase (LvECSOD) was cloned from the hepatopancreas of white shrimp Litopenaeus vannamei. It consisted of 915 bp nucleotides with an open reading frame corresponding to a deduced protein of 178 amino acids. The LvECSOD contains a putative signal peptide of 16 amino acids, two potential N-linked glycosylation sites (N(115)GTA and N(135)ITG) and a copper zinc superoxide dismutase family signature sequence (G(162)NAGaRvACctI(173)). It was found that four copper binding sites, four zinc binding sites and two cysteines involving in the formation of the disulfide bridge were conserved in the protein. LvECSOD shared 33-58% identity to ECSODs from other organisms. Expression analysis revealed that LvECSOD mRNA was widely distributed in all the tissues examined. When the shrimp challenged with Vibrio alginolyticus or white spot syndrome virus (WSSV), expression of LvECSOD mRNA in the hepatopancreas and hemocytes was mediated responsively. Our results suggested that LvECSOD was implicated in the immune response induced by V. alginolyticus and WSSV.     

Mj-DWD, a double WAP domain-containing protein with antiviral relevance in Marsupenaeus japonicus

The Mj-DWD (Marsupenaeus japonicus' double-WAP domains) gene was originally found up-regulated in virus-resistant shrimp M. japonicus by suppression subtractive hybridization (SSH). The full-length cDNA of Mj-DWD encodes a novel protein containing a KGD (Lys-Gly-Asp) motif and double WAP domains. Performed by quantitative real-time PCR, the expression level of Mj-DWD gene was consistently maintained at a high level in the newly prepared virus-resistant shrimp compared to the normal one. In addition, the Mj-DWD gene was also found to be rapidly up-regulated by WSSV infection during the early phase. Furthermore, the recombinant Mj-DWD, expressed by Pichia pastoris, showed specific protease inhibitory activity on Bacillus subtilis. These findings suggest that Mj-DWD plays an important role in the host defence system against WSSV infection in M. japonicus, possibly through its protease inhibitory activity.     

Molecular cloning and characterization of an antigenic protein with a repeating region from Clonorchis sinensis

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.     

RING-H2 protein WSSV249 from white spot syndrome virus sequesters a shrimp ubiquitin-conjugating enzyme, PvUbc, for viral pathogenesis

Modification of proteins by ubiquitin is essential for numerous cellular processes. The RING-H2 finger motif has been implicated in ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Four proteins, WSSV199, WSSV222, WSSV249, and WSSV403, from white spot syndrome virus (WSSV) contain the RING-H2 motif. Here we report that WSSV249 physically interacts with a shrimp ubiquitin-conjugating enzyme, PvUbc, and mediates ubiquitination through its RING-H2 motif in the presence of E1 and PvUbc. Mutations of the putative zinc coordination residues in the RING-H2 domain of WSSV249, however, ablate ubiquitination efficiency. In addition, the RING-H2 domain of WSSV249 is capable of ubiquitination with UbcH1, UbcH2, UbcH5a, UbcH5b, UbcH5c, UbcH6, and UbcH10, respectively, exhibiting a low degree of E2 specificity. Significantly, the expression of WSSV249 and PvUbc increased during infection, as revealed by real-time PCR. Furthermore, in situ hybridization showed that WSSV249 and PvUbc display similar expression patterns in infected shrimps, and immunofluorescence and immunohistochemistry assays showed an increase of PvUbc in infected shrimp cells. These results suggest that the RING-H2 protein WSSV249 from WSSV may function as an E3 ligase via sequestration of PvUbc for viral pathogenesis in shrimp.     

Molecular cloning and characterization of a putative protein kinase gene from the thermophilic fungus Thermomyces lanuginosus.

Based on the conserved amino acid sequence (DLKPEN) of serine-threonine protein kinase from several fungi, a degenerate primer was designed and synthesized. Total RNA was isolated from the thermophilic fungus Thermomyces lanuginosus. Using RACE-PCR, full-length cDNA of a putative serine-threonine protein kinase gene was cloned from T. lanuginosus. The full-length cDNA of T. lanuginosus protein kinase was 2551 bp and contained an 1806 bp open reading frame encoding a putative protein kinase precursor of 601 amino acid residues. Sequencing analysis showed that the cloned cDNA of T. lanuginosus had consensus protein kinase sequences. Conservative amino acid subdomains which most serine-threonine kinases contain can be found in the deduced amino acid sequence of T. lanuginosus putative protein kinase. Comparison results showed that the deduced amino acid sequence of T. lanuginosus putative protein kinase was highly homologous to that of Neurospora crassa dis1-suppressing protein kinase Dsk1. The putative protein kinase contained three arginine/serine-rich (SR) regions and two transmembrane domains. These showed that it might be a novel putative serine-threonine protein kinase.     

Shrimp vaccination trials with the VP292 protein of white spot syndrome virus

AIMS: Construction of a recombinant vector that expresses VP292 protein of white spot syndrome virus (WSSV) and to exploit the possibility of obtaining the vaccine conferring protection against WSSV infection in shrimps. METHODS AND RESULTS: VP292 protein of WSSV was amplified from WSSV genomic DNA by PCR. The target 814 bp amplified product specific for VP292 protein was inserted in to pQE30 expression vector. The recombinant plasmid of VP292 protein was transformed and expressed in Escherichia coli under induction of isopropyl-1-1-thio-beta-D-galactoside (IPTG) and the immunoreactivity of the fusion protein was detected by Western blot. Shrimp were vaccinated by intramuscular injection of the purified protein VP292 of WSSV and challenged for 0-30 days. Vaccination trial experiments show that two injections with recombinant VP292 (rVP292) protein induced a higher resistance, with 52% relative percentage survival value, in the shrimp at the 30th day postvaccination. CONCLUSIONS: The expression system of protein VP292 of WSSV with a high efficiency has been successfully constructed. Vaccination trials show significant resistance in the shrimp vaccinated twice with recombinant VP292. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study prosper the development of WSSV protein vaccine against WSSV infection in shrimps.     

The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases.

The primary structure of the 200 kDa protein of purified HeLa U5 snRNPs (U5-200kD) was characterized by cloning and sequencing of its cDNA. In order to confirm that U5-200kD is distinct from U5-220kD we demonstrate by protein sequencing that the human U5-specific 220 kDa protein is homologous to the yeast U5-specific protein Prp8p. A 246 kDa protein (Snu246p) homologous to U5-200kD was identified in Saccharomyces cerevisiae. Both proteins contain two conserved domains characteristic of the DEXH-box protein family of putative RNA helicases and RNA-stimulated ATPases. Antibodies raised against fusion proteins produced from fragments of the cloned mammalian cDNA interact specifically with the HeLa U5-200kD protein on Western blots and co-immunoprecipitate U5 snRNA and to a lesser extent U4 and U6 snRNAs from HeLa snRNPs. Similarly, U4, U5 and U6 snRNAs can be co-immunoprecipitated from yeast splicing extracts containing an HA-tagged derivative of Snu246p with HA-tag specific antibodies. U5-200kD and Snu246p are thus the first putative RNA helicases shown to be intrinsic components of snRNPs. Disruption of the SNU246 gene in yeast is lethal and leads to a splicing defect in vivo, indicating that the protein is essential for splicing. Anti-U5-200kD antibodies specifically block the second step of mammalian splicing in vitro, demonstrating for the first time that a DEXH-box protein is involved in mammalian splicing. We propose that U5-200kD and Snu246p promote one or more conformational changes in the dynamic network of RNA-RNA interactions in the spliceosome.