White Spot Syndrome Virus Structural Proteins Research Articles

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 analysis of the white spot syndrome virus major virion protein genes.

White spot syndrome virus (WSSV) is a member of a new virus family (Nimaviridae) infecting crustaceans. The regulation of transcription of WSSV genes is largely unknown. Transcription of the major WSSV structural virion protein genes, vp28, vp26, vp24, vp19 and vp15, was studied to search for common promoter motifs for coordinate expression. The temporal expression of these genes and both 5' and 3' ends of the mRNA were determined, using infected crayfish gill tissue as a RNA source. RT-PCR showed that all five genes are expressed late in infection compared to the early ribonucleotide reductase large subunit gene. 5' RACE studies revealed a consensus late transcription initiation motif for only two of the five major virion protein genes. This motif was only found in one other upstream region of the putative translational start site of a gene with unknown function (ORF 158). No other conserved sequence motifs could be detected in the sequences surrounding the transcriptional start sites of the five major virion protein genes. All 5' ends were located about 25 nt downstream of an A/T rich sequence, including the consensus TATA-box sequence for vp15. The absence of a consensus motif is distinct from gene regulation of other large dsDNA viruses and suggests a unique regulation of WSSV transcription, in line with its unique taxonomic position.

Characterization of a novel envelope protein (VP281) of shrimp white spot syndrome virus by mass spectrometry.

The primary structure of a novel envelope protein from shrimp white spot syndrome virus (WSSV) was characterized using a combination of SDS-PAGE and mass spectrometry. The resulting amino acid sequence matched an open reading frame (ORF), ORF1050, of the WSSV genome ORF database. ORF1050 contained 843 nt, encoding 281 aa, and was termed the vp281 gene. Computer-assisted analysis showed that both the vp281 gene and its product shared no significant homology with other known viruses. However, they shared striking identity/similarity with another WSSV structural protein, VP292, at both the nucleotide and amino acid sequence level, suggesting that vp281 and vp292 might have evolved by gene duplication from a common ancestral gene. WSSV VP281 cDNA was cloned into a pET32a(+) expression vector containing a T7 RNA polymerase promoter to produce (His)(6)-tagged fusion proteins in Escherichia coli strain BL21. Specific mouse antibodies were raised using the purified fusion protein (His)(6)-VP281. Western blot analysis showed that the mouse anti-(His)(6)-VP281 antibodies bound specifically to VP281 of WSSV, without cross-reactivity with VP292. The transmission electron microscope immunogold-labelling method was used to localize VP281 in the WSSV virion as an envelope protein. The cell attachment 'Arg-Gly-Asp' motif in VP281 indicated that this protein might play an important role in mediating WSSV infectivity.

Production of polyclonal antiserum specific to the 27.5 kDa envelope protein of white spot syndrome virus.

A truncated version of the white spot syndrome virus (WSSV) 27.5 kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli. The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture. Antiserum from a rabbit immunized with the recombinant protein recognized the 27.5 kDa viral envelope protein of WSSV isolated from different geographic regions. The antiserum did not recognize any of the other known WSSV structural proteins. A sensitive immunodot assay for WSSV was developed using the specific rabbit polyclonal antiserum.