Jiann Horng Leu

Genomic and Proteomic Analysis of Thirty-Nine Structural Proteins of Shrimp White Spot Syndrome Virus

ABSTRACT 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.

Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion.

ABSTRACT The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.

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

BackgroundWhite 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.ResultsSequencing 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/.ConclusionThis 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.

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.

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

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.

Shrimp Pm-fortilin inhibits the expression of early and late genes of white spot syndrome virus (WSSV) in an insect cell model

Fortilin plays an important role in anti-apoptotic mechanisms and cell proliferation in many eukaryotic organisms. This work confirmed previous reports that Sf9 can support the replication of white spot syndrome virus (WSSV) genomic material by using immunohistochemistry with a specific antibody to detect the immediate early gene 1 (ie1) and by amplification of WSSV DNA and mRNA products. Using this insect-cell model system, we show that overexpression of Pm-fortilin in Sf9 cells inhibited the expression of WSSV early genes and late genes (WSSV-DNA polymerase, VP15 and VP28) but not an immediate early gene ie1. This is the first time that an insect cell line has been used to demonstrate interaction between a shrimp gene and genes of a shrimp virus.

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

White spot syndrome virus (WSSV) is a large (≈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.

A Review of the Major Penaeid Shrimp EST Studies and the Construction of a Shrimp Transcriptome Database Based on the ESTs from Four Penaeid Shrimp

By economic value, shrimp is currently the most important seafood commodity worldwide, and these animals are often the subject of scientific research in shrimp farming countries. High throughput methods, such as expressed sequence tags (ESTs), were originally developed to study human genomics, but they are now available for studying other important organisms, including shrimp. ESTs are short sequences generated by sequencing randomly selected cDNA clones from a cDNA library. This is currently the most efficient and powerful method for providing transcriptomic data for organisms with an uncharacterized genome. This review will summarize the sixteen major shrimp EST studies that have been conducted to date. In addition, we analyzed the EST data downloaded from NCBI dbEST for the four major penaeid shrimp species and constructed a database to host all of these EST data as well as our own analysis results. This database provides the shrimp aquaculture research community with an outline of the shrimp transcriptome as well as a tool for shrimp gene identification.

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

ABSTRACT 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.

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.