Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.

Although the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is part of the antiviral response in arthropods such as Drosophila, here we show that white spot syndrome virus (WSSV) uses a shrimp STAT as a transcription factor to enhance viral gene expression in host cells. In a series of deletion and mutation assays using the WSSV immediate-early gene ie1 promoter, which is active in shrimp cells and also in insect Sf9 cells, an element containing a STAT binding motif was shown to be important for the overall level of WSSV ie1 promoter activity. In the Sf9 insect cell line, a specific protein-DNA complex was detected by using electrophoresis mobility shift assays (EMSA) with the 32 P-labeled STAT binding motif of the WSSV ie1 promoter as the probe. When recombinant Penaeus monodon STAT (rPmSTAT) was overexpressed in Sf9 cells, EMSA with specific antibodies confirmed that the STAT was responsible for the formation of the specific protein-DNA complex. Another EMSA showed that in WSSV-infected P. monodon, levels of activated PmSTAT were higher than in WSSV-free P. monodon. A transactivation assay of the WSSV ie1 promoter demonstrated that increasing the level of rPmSTAT led to dose-dependent increases in ie1 promoter activity. These results show that STAT directly transactivates WSSV ie1 gene expression and contributes to its high promoter activity. We conclude that WSSV successfully annexes a putative shrimp defense mechanism, which it uses to enhance the expression of viral immediate-early genes.

Immediate-early proteins from many viruses function as transcriptional regulators and exhibit transactivation activity, DNA binding activity, and dimerization. In this study, we investigated these characteristics in white spot syndrome virus (WSSV) immediate-early protein 1 (IE1) and attempted to map the corresponding functional domains. Transactivation was investigated by transiently expressing a protein consisting of the DNA binding domain of the yeast transactivator GAL4 fused to full-length IE1. This GAL4-IE1 fusion protein successfully activated the Autographa californica multicapsid nucleopolyhedrovirus p35 basal promoter when five copies of the GAL4 DNA binding site were inserted upstream of the TATA box. A deletion series of GAL4-IE1 fusion proteins suggested that the transactivation domain of WSSV IE1 was carried within its first 80 amino acids. A point mutation assay further showed that all 12 of the acidic residues in this highly acidic domain were important for IE1's transactivation activity. DNA binding activity was confirmed by an electrophoresis mobility shift assay using a probe with 32 P-labeled random oligonucleotides. The DNA binding region of WSSV IE1 was located in its C-terminal end (amino acids 81 to 224), but mutation of a putative zinc finger motif in this C-terminal region suggested that this motif was not directly involved in the DNA binding activity. A homotypic interaction between IE1 molecules was demonstrated by glutathione S-transferase pull-down assay and a coimmunoprecipitation analysis. A glutaraldehyde cross-linking experiment and gel filtration analysis showed that this self-interaction led to the formation of stable IE1 dimers.White spot syndrome virus (WSSV) is the causative agent of a disease that has led to severe mortalities of cultured shrimps all over the world (10,14,23,53). WSSV is a large doublestranded DNA virus which is extremely virulent (23,38,39), has a wide host range (14, 33), and targets various tissues (32, 59). It was recently erected as the type species of genus Whispovirus in the family Nimaviridae (56). Although the complete sequence of the WSSV genome has been known for several years (7,55,60), knowledge of the biological functions of the viral proteins is still quite poor. The WSSV immediate-early gene ie1 (31) was recently shown to use a shrimp signal transducer and activator of transcription (STAT) as a transcription factor to enhance its expression and contribute to its high promoter activity in host cells (30). In the present study, we further investigate the characteristics of WSSV IE1. This is made more difficult by the fact that no continuous shrimp cell line is currently available, and while bearing in mind that a heterologous system might introduce experimental artifacts, here we follow previous studies (22,30,34) and use the Sf9 insect cell system. Many viral immediate-early genes encode multifunctional transcriptional regulators that both positively and negatively modulate gene expression (26,52,57). These transcriptional regulators must po...

In this study, we characterize a novel white spot syndrome virus (WSSV) structural protein, VP51A (WSSV-TW open reading frame 294), identified from a previous mass spectrometry study. Temporal-transcription analysis showed that vp51A is expressed in the late stage of WSSV infection. Gene structure analysis showed that the transcription initiation site of vp51A was 135 bp upstream of the translation start codon. The poly(A) addition signal overlapped with the translation stop codon, TAA, and the poly(A) tail was 23 bp downstream of the TAA. Western blot analysis of viral protein fractions and immunoelectron microscopy both suggested that VP51A is a viral envelope protein. Western blotting of the total proteins extracted from WSSV virions detected a band that was close to the predicted 51-kDa mass, but the strongest signal was around 72 kDa. We concluded that this 72-kDa band was in fact the full-length VP51A protein. Membrane topology assays demonstrated that the VP51A 72-kDa protein is a type II transmembrane protein with a highly hydrophobic transmembrane domain on its N terminus and a C terminus that is exposed on the surface of the virion. Coimmunoprecipitation, colocalization, and yeast two-hybrid assays revealed that VP51A associated directly with VP26 and indirectly with VP28, with VP26 acting as a linker protein in the formation of a VP51A-VP26-VP28 complex.Viral structural proteins, especially the envelope proteins, are important, not only because they are involved in virion morphogenesis, but also because they are the first molecules to interact with the host. The structural proteins often play vital roles in cell targeting, virus entry, assembly, and budding (1, 2, 21, 22, 24), as well as triggering host antiviral defenses (26). In the case of white spot syndrome virus (WSSV) (genus Whispovirus, family Nimaviridae) (37), a double-stranded DNA virus that has caused severe mortality and huge economic losses to the shrimp farming industry globally for more than a decade (5, 19), proteomic methods have helped to identify a total of 58 structural proteins, over 30 of which are currently recognized as envelope proteins (13,31,44,47). Some of the WSSV envelope proteins involved in shrimp infection have been identified (12,14,34,36,41,43), and these envelope and other WSSV structural proteins have been used in various studies, including RNA interference-based gene knockdown to silence viral structural-protein gene expression (8, 27, 45), DNA and protein vaccination to elevate host immunity (25,29,36,39), and antibody neutralization techniques that neutralize the virus by preventing envelope proteins from interacting with host cell receptors (12,34,41,43).In the present paper, we characterize and investigate the functionality of a WSSV structural protein that was first reported by Tsai et al. (32). This protein, designated VP51A, corresponds to open reading frame 294 of the WSSV-TW isolate, and its gene encodes a polypeptide of 486 amino acids (aa) with a theoretical mass of 51 kDa. A method was recently establ...

Here, we investigate the roles of copepods and bivalve mollusks in the transmission of white spot syndrome virus (WSSV), which is the causative pathogen of an acute, contagious disease that causes severe mortalities in cultured shrimp. Copepods are common components in seawater ponds and are often eaten as live food by shrimp post-larvae. WSSV has been detected in these animals, but it is unknown whether this was due to contamination or infection. Meanwhile, the bivalve mollusk Meretrix lusoria is often used as live food for brooders, and in Taiwan, this hard clam is sometimes co-cultured with shrimp in farming ponds. However, mollusks' ability to accumulate, or allow the replication of, shrimp viruses has not previously been studied. In this study, WSSV, the copepod Apocyclops royi and bivalve mollusk M. lusoria were experimentally challenged with WSSV and then assayed for both the presence of the virus and for viral gene expression. Results showed that the WSSV genome could be detected and that the viral loads were increased in a time-dependent manner after challenge both in A. royi and M. lusoria. Reverse transcriptase PCR monitoring of WSSV gene expression showed that WSSV could replicate in A. royi but not in M. lusoria, which suggested that WSSV, while could infect A. royi, was only accumulated in M. lusoria. A bioassay further showed that the WSSV accumulated in M. lusoria could be transmitted to Litopenaeus vannamei and cause severe infection.