A bacterial binary toxin system that kills both insects and aquatic crustaceans: Photorhabdus insect-related toxins A and B.

Photorhabdus insect-related toxins A and B (PirA and PirB) were first recognized as insecticidal toxins from Photorhabdus luminescens. However, subsequent studies showed that their homologs from Vibrio parahaemolyticus also play critical roles in the pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimps. Based on the structural features of the PirA/PirB toxins, it was suggested that they might function in the same way as a Bacillus thuringiensis Cry pore-forming toxin. However, unlike Cry toxins, studies on the PirA/PirB toxins are still scarce, and their cytotoxic mechanism remains to be clarified. In this review, based on our studies of V. parahaemolyticus PirAvp/PirBvp, we summarize the current understanding of the gene locations, expression control, activation, and cytotoxic mechanism of this type of toxin. Given the important role these toxins play in aquatic disease and their potential use in pest control applications, we also suggest further topics for research. We hope the information presented here will be helpful for future PirA/PirB studies.

Shrimp SIRT1 activates of the WSSV IE1 promoter independently of the NF-κB binding site.

Since the mechanisms by which cellular factors modulate replication of the shrimp viral pathogen white spot syndrome virus (WSSV) are still largely unknown, here we consider the sirtuins, a family of NAD+-dependent protein deacetylases that are known to function as regulatory factors that activate or suppress viral transcription and replication in mammals. In particular, we focus on SIRT1 by isolating and characterizing LvSIRT1 from white shrimp (Litopenaeus vannamei) and investigating its involvement in WSSV infection. DsRNA-mediated gene silencing led to the expression of WSSV genes and the replication of genomic DNAs being significantly decreased in LvSIRT1-silenced shrimp. The deacetylase activity of LvSIRT1 was significantly induced at the early stage (2 hpi) and the genome replication stage (12 hpi) of WSSV replication, but decreased at the late stage of WSSV replication (24 hpi). Treatment with the SIRT1 activator resveratrol further suggested that LvSIRT1 activation increased the expression of several WSSV genes (IE1, VP28 and ICP11). Lastly, we used transfection and dual luciferase assays in Sf9 insect cells to show that while the overexpression of LvSIRT1 facilitates the promoter activity of WSSV IE1, this enhancement of WSSV IE1 expression is achieved by a transactivation pathway that is NF-κB-independent.

The genome and occlusion bodies of marine Penaeus monodon nudivirus (PmNV, also known as MBV and PemoNPV) suggest that it should be assigned to a new nudivirus genus that is distinct from the terrestrial nudiviruses.

BACKGROUND: Penaeus monodon nudivirus (PmNV) is the causative agent of spherical baculovirosis in shrimp (Penaeus monodon). This disease causes significant mortalities at the larval stage and early postlarval (PL) stage and may suppress growth and reduce survival and production in aquaculture. The nomenclature and classification status of PmNV has been changed several times due to morphological observation and phylogenetic analysis of its partial genome sequence. In this study, we therefore completed the genome sequence and constructed phylogenetic trees to clarify PmNV's taxonomic position. To better understand the characteristics of the occlusion bodies formed by this marine occluded virus, we also compared the chemical properties of the polyhedrin produced by PmNV and the baculovirus AcMNPV (Autographa californica nucleopolyhedrovirus). RESULTS: We used next generation sequencing and traditional PCR methods to obtain the complete PmNV genome sequence of 119,638 bp encoding 115 putative ORFs. Phylogenetic tree analysis showed that several PmNV genes and sequences clustered with the non-occluded nudiviruses and not with the baculoviruses. We also investigated the characteristics of PmNV polyhedrin, which is a functionally important protein and the major component of the viral OBs (occlusion bodies). We found that both recombinant PmNV polyhedrin and wild-type PmNV OBs were sensitive to acid conditions, but unlike the baculoviral OBs, they were not susceptible to alkali treatment. CONCLUSIONS: From the viral genome features and phylogenetic analysis we conclude that PmNV is not a baculovirus, and that it should be assigned to the proposed Nudiviridae family with the other nudiviruses, but into a distinct new genus (Gammanudivirus).

A newly identified protein complex that mediates white spot syndrome virus infection via chitin-binding protein.

White spot syndrome virus (WSSV) is a large enveloped virus which has caused severe mortality and huge economic losses in the shrimp farming industry. The enveloped virus must be combined with the receptors of the host cell membrane by the virus envelope proteins. In the case of WSSV, binding of envelope proteins with receptors of the host cell membrane was discovered in a number of previous studies, such as VP53A and 10 other proteins with chitin-binding protein (CBP), VP28 with Penaeus monodon Rab7, VP187 with ß-integrin, and so on. WSSV envelope proteins were also considered capable of forming a protein complex dubbed an 'infectome'. In this study, the research was focused on the role of CBP in the WSSV infection process, and the relationship between CBP and the envelope proteins VP24, VP28, VP31, VP32 VP39B, VP53A and VP56. The results of the reverse transcription-PCR analyses showed that CBP existed in a variety of shrimp. The speed of WSSV infection could be slowed down by inhibiting CBP gene expression. Far-Western blot analysis and His pull-down assays were conducted, and a protein complex was found that appeared to be composed of a 'linker' protein consisting of VP31, VP32 and VP39B together with four envelope proteins, including VP24, VP28, VP53A and VP56. This protein complex was possibly another part of the infectome and the possible binding region with CBP. The findings of this study may have identified certain points for further WSSV research.

Endocytic pathway is indicated for white spot syndrome virus (WSSV) entry in shrimp.

The white spot syndrome virus (WSSV) has had a serious economic impact on the global shrimp aquaculture industry in the past two decades. Although research has clarified a lot about its genome and structure, the mechanism of how WSSV enters a cell is still unclear. In this study to determine this mechanism, primary cultured hemocytes were used as an experimental model to observe the process of WSSV entry because the stable shrimp cell lines for WSSV infection are lacking. After labeling virions and endosomes with fluorescent dyes followed by observation with a confocal microscope, the results show that the WSSV colocalizes with early endosomes. Hemocytes are further treated with different endocytic inhibitors, methyl-ß-cyclodextrin (MßCD) and chlorpromazine (CPZ). WSSV still can be detected in the hemocytes treated with CPZ, but not in the hemocytes treated with MßCD. Thus, we conclude that WSSV adopts the caveolae-mediated endocytosis to enter the shrimp cell.

A model for apoptotic interaction between white spot syndrome virus and shrimp.

White spot syndrome virus (WSSV) is an enveloped, large dsDNA virus that mainly infects penaeid shrimp, causing serious damage to the shrimp aquaculture industry. Like other animal viruses, WSSV infection induces apoptosis. Although this occurs even in by-stander cells that are free of WSSV virions, apoptosis is generally regarded as a kind of antiviral immune response. To counter this response, WSSV has evolved several different strategies. From the presently available literature, we construct a model of how the host and virus both attempt to regulate apoptosis to their respective advantage. The basic sequence of events is as follows: first, when a WSSV infection occurs, cellular sensors detect the invading virus, and activate signaling pathways that lead to (1) the expression of pro-apoptosis proteins, including PmCasp (an effecter caspase), MjCaspase (an initiator caspase) and voltage-dependent anion channel (VDAC); and (2) mitochondrial changes, including the induction of mitochondrial membrane permeabilization and increased oxidative stress. These events initiate the apoptosis program. Meanwhile, WSSV begins to express its genes, including two anti-apoptosis proteins: AAP-1, which is a direct caspase inhibitor, and WSV222, which is an E3 ubiquitin ligase that blocks apoptosis through the ubiquitin-mediated degradation of shrimp TSL protein (an apoptosis inducer). WSSV also induces the expression of a shrimp anti-apoptosis protein, Pm-fortilin, which can act on Bax to inhibit mitochondria-triggered apoptosis. This is a life and death struggle because the virus needs to prevent apoptosis in order to replicate. If WSSV succeeds in replicating in sufficient numbers, this will result in the death of the infected penaeid shrimp host.

The role of aldehyde dehydrogenase and hsp70 in suppression of white spot syndrome virus replication at high temperature.

High temperature (32 to 33°C) has been shown to reduce mortality in white spot syndrome virus (WSSV)-infected shrimps, but the mechanism still remains unclear. Here we show that in WSSV-infected shrimps cultured at 32°C, transcriptional levels of representative immediate-early, early, and late genes were initially higher than those at 25°C. However, neither the IE1 nor VP28 protein was detected at 32°C, suggesting that high temperature might inhibit WSSV protein synthesis. Two-dimensional gel electrophoresis analysis revealed two proteins, NAD-dependent aldehyde dehydrogenase (ALDH) and the proteasome alpha 4 subunit (proteasome α4), that were markedly upregulated in WSSV-infected shrimps at 32°C. Reverse transcription-PCR (RT-PCR) analysis of members of the heat shock protein family also showed that hsp70 was upregulated at 32°C. When aldh, proteasome α4, and hsp70 were knocked down by double-stranded RNA interference and shrimps were challenged with WSSV, the aldh and hsp70 knockdown shrimps became severely infected at 32°C, while the proteasome α4 knockdown shrimps remained uninfected. Our results therefore suggest that ALDH and Hsp70 both play an important role in the inhibition of WSSV replication at high temperature.

Differential expression of LvHSP60 in shrimp in response to environmental stress.

Previous studies showed that heat-shock protein 60 (HSP60) was known to function as a molecular chaperone and is an important factor in the innate immune system in mammals. However, little was known about the physiological relevance of HSP60 in marine invertebrates. This study focuses on long-term monitoring of the differential expression of LvHSP60 in shrimp Litopenaeus vannamei in response to environmental stress. The thermal aggregation assay elucidated that LvHSP60 was an effective chaperone. It also suggested that LvHSP60 may employ the cell's intrinsic mechanism to start the immunizing process. Using quantitative real-time PCR to monitor gene expression showed that LvHSP60 was variable under different stresses including environmental stress and pathogenic infection. LvHSP60 was speculated to regulate the adaptive responses to overcome environmental stresses. In conclusion, our study proved that LvHSP60 plays an important role in the intrinsic immune system and stress responses of shrimp.

White spot syndrome virus protein ICP11: A histone-binding DNA mimic that disrupts nucleosome assembly.

White spot syndrome virus (WSSV) is a large ( approximately 300 kbp), double-stranded DNA eukaryotic virus that has caused serious disease in crustaceans worldwide. ICP11 is the most highly expressed WSSV nonstructural gene/protein, which strongly suggests its importance in WSSV infection; but until now, its function has remained obscure. We show here that ICP11 acts as a DNA mimic. In crystal, ICP11 formed a polymer of dimers with 2 rows of negatively charged spots that approximated the duplex arrangement of the phosphate groups in DNA. Functionally, ICP11 prevented DNA from binding to histone proteins H2A, H2B, H3, and H2A.x, and in hemocytes from WSSV-infected shrimp, ICP11 colocalized with histone H3 and activated-H2A.x. These observations together suggest that ICP11 might interfere with nucleosome assembly and prevent H2A.x from fulfilling its critical function of repairing DNA double strand breaks. Therefore, ICP11 possesses a functionality that is unique among the handful of presently known DNA mimic proteins.

Feasibility Study on the Use of Fly Maggots (Musca domestica) as Carriers to Inhibit Shrimp White Spot Syndrome.

The shrimp aquaculture industry has encountered many diseases that have caused significant losses, with the most serious being white spot syndrome (WSS). Until now, no cures, vaccines, or drugs have been found to counteract the WSS virus (WSSV). The purpose of this study was to develop an oral delivery system to transport recombinant proteinaceous antigens into shrimp. To evaluate the feasibility of the oral delivery system, we used white shrimp as the test species and maggots as protein carriers. The results indicated that the target protein was successfully preserved in the maggot, and the protein was detected in the gastrointestinal tract of the shrimp, showing that this oral delivery system could deliver the target protein to the shrimp intestine, where it was absorbed. In addition, the maggots were found to increase the total haemocyte count and phenoloxidase activity of the shrimp, and feeding shrimp rVP24-fed maggots significantly induced the expression of penaeidins 2. In the WSSV challenge, the survival rate of rVP24-fed maggots was approximately 43%. This study showed that maggots can be used as effective oral delivery systems for aquatic products and may provide a new method for aquatic vaccine delivery systems.

Identification and characterization of l-amino acid oxidase 2 gene in orange-spotted grouper (Epinephelus coioides).

Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens.

Transactivation, dimerization, and DNA-binding activity of white spot syndrome virus immediate-early protein IE1.

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.

Identification and expression analysis of 19 CC chemokine genes in orange-spotted grouper (Epinephelus coioides).

In this study, we describe 19 different CC chemokine genes from the orange-spotted grouper, Epinephelus coioides, identified by the analysis of the spleen transcriptome. Multiple sequence alignment of the 19 CC chemokines showed that although two genes, EcSCYA115 and EcSCYA117, shared 80% amino acid similarity (72% identity), the majority exhibited low similarity to each other. Phylogenetic analysis divided the 19 CC chemokines into six major groups. Tissue distribution analysis by RT-PCR showed that most of these chemokines were ubiquitously expressed in the 9 examined tissues, whereas some exhibited tissue-preferential expression patterns. For example, EcSCYA103 was preferentially expressed in fin and gill; EcSCYA109 in head kidney and spleen; EcSCYA114 in fin, gill, and liver; and EcSCYA119 in fin and stomach. Quantitative RT-PCR showed that after challenge with grouper iridovirus (GIV), four of the 19 CC chemokine genes, EcSYCA102, EcSYCA103, EcSYCA116, and EcSYCA118, were highly induced in the spleen. The expression of these four genes could also be upregulated by LPS and poly (I:C) challenges, suggesting that these four genes might be involved in immune response against invading pathogens.

Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon.

The inhibitor of apoptosis proteins (IAPs) play important roles in both apoptosis and innate immunity. Here, we report the first cloning and characterization of a novel IAP family member, PmIAP, from Penaeus monodon. The full-length PmIAP cDNA is 4769bp, with an ORF encoding a protein of 698 amino acids. The PmIAP protein contains three BIR domains and a C-terminal RING domain, and its mRNA was expressed in all analyzed tissues. In insect cells, PmIAP, together with Spodoptera frugiperda IAP, AcMNPV P35, and WSSV449 (or ORF390, an anti-apoptosis protein encoded by white spot syndrome virus), could all block the apoptosis induced by Drosophila Reaper protein (Rpr), whereas only P35 and WSSV449 could block the apoptosis induced by actinomycin D. Co-immunoprecipitation showed that PmIAP physically interacted with Rpr, and in an immunofluorescent analysis the two proteins produced co-localized punctate signals in the cytoplasm. Deletion analysis revealed that both the BIR2 and BIR3 domains of PmIAP could independently bind to and inhibit Rpr, whereas the BIR1 domain could not. These results strongly suggest that PmIAP blocks Rpr's pro-apoptotic activity through mechanisms that are evolutionarily conserved across crustaceans, insects, and mammals.

Penaeus monodon caspase is targeted by a white spot syndrome virus anti-apoptosis protein.

Caspases play a central and evolutionarily conserved role in mediating and executing apoptosis. Here, we report the cloning and characterization of a caspase from Penaeus monodon, Pm caspase. The full-length Pm caspase cDNA is 1386bp, encoding a polypeptide of 304 amino acids with a calculated molecular mass of 34.3kDa. BLASTP analysis against the NCBI nr database showed that Pm caspase is similar to insect effector caspases. RT-PCR analysis showed that Pm caspase mRNA is expressed in all examined tissues. When Pm caspase was overexpressed in SF-9 cells, the cells showed apoptotic morphological features, including the formation of apoptotic bodies and DNA ladders. The caspase-3 activity of Pm caspase was determined using the recombinant protein purified from Escherichia coli. Both RT-PCR and qRT-PCR analyses showed that the RNA levels of Pm caspase and P. monodon inhibitor of apoptosis protein (PmIAP) remained unchanged after white spot syndrome virus (WSSV) infection. We also used Pm caspase to show that WSSV449, an anti-apoptosis protein encoded by WSSV, is a direct caspase inhibitor.

WSSV infection activates STAT in shrimp.

Although the JAK/STAT signaling pathway is usually involved in antiviral defense, a recent study suggested that STAT might be annexed by WSSV (white spot syndrome virus) to enhance the expression of a viral immediate early gene in infected shrimps. In the present study, we clone and report the first full-length cDNA sequence for a crustacean STAT from Penaeus monodon. Alignment and comparison with the deduced amino acid sequences of other STATs identified several important conserved residues and functional domains, including the DNA binding domain, SH2 domain and C-terminal transactivation domain. Based on these conserved sequences, a phylogenetic analysis suggested that shrimp STAT belongs to the ancient STAT family, while the presence of the functional domains suggested that shrimp STAT might share similar functions and regulating mechanisms with the well-known STATs isolated from model organisms. Real-time PCR showed a decreased transcription level of shrimp STAT after WSSV infection, but a Western blot analysis using anti-phosphorylated STAT antibody showed an increased level of phosphorylated (activated) STAT in the lymphoid organ of shrimp after WSSV infection. We further show that a primary culture of lymphoid organ cells from WSSV-infected shrimp resulted in activated STAT being translocated from the cytoplasm to the nucleus. This report provides experimental evidence that shrimp STAT is activated in response to WSSV infection. Our results support an earlier finding that WSSV does not disrupt JAK/STAT pathway, but on the contrary benefits from STAT activation in the shrimp host.

Comparative analysis of differentially expressed genes in normal and white spot syndrome virus infected Penaeus monodon.

BACKGROUND: White spot syndrome (WSS) is a viral disease that affects most of the commercially important shrimps and causes serious economic losses to the shrimp farming industry worldwide. However, little information is available in terms of the molecular mechanisms of the host-virus interaction. In this study, we used an expressed sequence tag (EST) approach to observe global gene expression changes in white spot syndrome virus (WSSV)-infected postlarvae of Penaeus monodon. RESULTS: Sequencing of the complementary DNA clones of two libraries constructed from normal and WSSV-infected postlarvae produced a total of 15,981 high-quality ESTs. Of these ESTs, 46% were successfully matched against annotated genes in National Center of Biotechnology Information (NCBI) non-redundant (nr) database and 44% were functionally classified using the Gene Ontology (GO) scheme. Comparative EST analyses suggested that, in postlarval shrimp, WSSV infection strongly modulates the gene expression patterns in several organs or tissues, including the hepatopancreas, muscle, eyestalk and cuticle. Our data suggest that several basic cellular metabolic processes are likely to be affected, including oxidative phosphorylation, protein synthesis, the glycolytic pathway, and calcium ion balance. A group of immune-related chitin-binding protein genes is also likely to be strongly up regulated after WSSV infection. A database containing all the sequence data and analysis results is accessible at http://xbio.lifescience.ntu.edu.tw/pm/. CONCLUSION: This study suggests that WSSV infection modulates expression of various kinds of genes. The predicted gene expression pattern changes not only reflect the possible responses of shrimp to the virus infection but also suggest how WSSV subverts cellular functions for virus multiplication. In addition, the ESTs reported in this study provide a rich source for identification of novel genes in shrimp.

Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection.

To better understand the pathogenesis of white spot syndrome virus (WSSV) and to determine which cell pathways might be affected after WSSV infection, two-dimensional gel electrophoresis (2-DE) was used to produce protein expression profiles from samples taken at 48 h post-infection (hpi) from the stomachs of Litopenaeus vannamei (also called Penaeus vannamei) that were either specific pathogen free or else infected with WSSV. Seventy-five protein spots that consistently showed either a marked change (>50%) in accumulated levels or else were highly expressed throughout the course of WSSV infection were selected for further study. After in-gel trypsin digestion followed by LC-nanoESI-MS/MS, bioinformatics databases were searched for matches. A total of 53 proteins were identified, with functions that included energy production, calcium homeostasis, nucleic acid synthesis, signaling/communication, oxygen carrier/transportation, and SUMO-related modification. 2-DE results were shown to be consistent with relative EST database data from a previously developed EST database of two Penaeus monodon cDNA libraries. For seven selected genes, 2-DE and EST data were also compared with transcriptional time-course RT-PCR data. This study is the first global analysis of differentially expressed proteins in WSSV-infected shrimp, and in addition to increasing our understanding of the molecular pathogenesis of this virus-associated shrimp disease, the results presented here should be useful both for identifying potential biomarkers and for developing antiviral measures.

The novel white spot syndrome virus-induced gene, PmERP15, encodes an ER stress-responsive protein in black tiger shrimp, Penaeus monodon.

By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54-59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp.

The promoter of the white spot syndrome virus immediate-early gene WSSV108 is activated by the cellular KLF transcription factor.

A series of deletion and mutation assays of the white spot syndrome virus (WSSV) immediate-early gene WSSV108 promoter showed that a Krüppel-like factor (KLF) binding site located from -504 to -495 (relative to the transcription start site) is important for the overall level of WSSV108 promoter activity. Electrophoretic mobility shift assays further showed that overexpressed recombinant Penaeus monodon KLF (rPmKLF) formed a specific protein-DNA complex with the (32)P-labeled KLF binding site of the WSSV108 promoter, and that higher levels of Litopenaeus vannamei KLF (LvKLF) were expressed in WSSV-infected shrimp. A transactivation assay indicated that the WSSV108 promoter was strongly activated by rPmKLF in a dose-dependent manner. Lastly, we found that specific silencing of LvKLF expression in vivo by dsRNA injection dramatically reduced both WSSV108 expression and WSSV replication. We conclude that shrimp KLF is important for WSSV genome replication and gene expression, and that it binds to the WSSV108 promoter to enhance the expression of this immediate-early gene.