Shrimp miR-10a Is Co-opted by White Spot Syndrome Virus to Increase Viral Gene Expression and Viral Replication.

Members of the microRNA miR-10 family are highly conserved and play many important roles in diverse biological mechanisms, including immune-related responses and cancer-related processes in certain types of cancer. In this study, we found the most highly upregulated shrimp microRNA from Penaeus vannamei during white spot syndrome virus (WSSV) infection was miR-10a. After confirming the expression level of miR-10a by northern blot and quantitative RT-PCR, an in vivo experiment showed that the viral copy number was decreased in miR-10a-inhibited shrimp. We found that miR-10a targeted the 5' untranslated region (UTR) of at least three viral genes (vp26, vp28, and wssv102), and plasmids that were controlled by the 5' UTR of these genes produced enhanced luciferase signals in transfected SF9 cells. These results suggest a previously unreported role for shrimp miR-10a and even a new type of host-virus interaction, whereby a co-opts the key cellular regulator miR-10a to globally enhance the translation of viral proteins.

Six Hours after Infection, the Metabolic Changes Induced by WSSV Neutralize the Host's Oxidative Stress Defenses.

Levels of intracellular ROS (reactive oxygen species) were significantly increased in hemocytes collected from WSSV-infected shrimp within the first 30-120 min after infection. Measurement of the NADPH/NADP(+) and GSH/GSSG ratios revealed that after a significant imbalance toward the oxidized forms at 2 hpi, redox equilibrium was subsequently restored. Meanwhile, high levels of lactic acid production, elevated NADH/NAD(+) ratios, and metabolic changes in the glycolysis pathway show that the Warburg effect was triggered by the virus. The timing of these changes suggests that WSSV uses this metabolic shift into aerobic glycolysis to counteract the high levels of ROS produced in response to viral infection. We further show that if the Warburg effect is inhibited by chemical inhibition of the PI3K-Akt-mTOR signaling pathway, or if the pentose phosphate pathway is chemically inhibited, then in both cases, the production of intracellular ROS is sustained. We conclude that WSSV uses the PI3K-Akt-mTOR-regulated Warburg effect to restore host redox balance and to counter the ROS produced by the host in response to WSSV infection. We also found that pyruvate kinase activity was inhibited by WSSV. This inhibition is likely to increase the availability of the raw materials essential for WSSV gene expression and replication.

Laminin Receptor in Shrimp Is a Cellular Attachment Receptor for White Spot Syndrome Virus.

White spot syndrome virus (WSSV, genus Whispovirus, family Nimaviridae) is causing huge economic losses in global shrimp farming, but there is no effective control. Shrimp cell laminin receptor (Lamr) may have a role in WSSV infection. The objective was to characterize interactions between Penaeus monodon Lamr (PmLamr) and WSSV structural proteins. In this study, PmLamr interacted with nine WSSV structural proteins (based on yeast two-hybrid screening), of which one (VP31) was characterized. Protein pull-down assay confirmed the interaction between PmLamr and VP31; the latter was an envelope protein exposed outside the WSSV virion (based on membrane topology assays). Furthermore, similar to mammalian Lamr, there were two major protein bands in shrimp cells. Cellular localization assay demonstrated VP31 co-localized with PmLamr on transfected cells. Enzyme-link immunosorbent assay (ELISA) and competitive ELISA demonstrated binding of VP31 on PmLamr was dose-dependent; however, addition of WSSV virion competed for binding affinity. Furthermore, based on an in vivo neutralization assay, both VP31 and PmLamr delayed mortality in shrimp challenged with WSSV. We concluded Lamr was an important receptor for WSSV infection and the viral envelope protein VP31 may have a role in host cell recognition and binding. These data contributed to elucidating pathogenesis of WSSV infection and may help in controlling this disease.

White spot syndrome virus protein kinase 1 defeats the host cell's iron-withholding defense mechanism by interacting with host ferritin.

UNLABELLED: Iron is an essential nutrient for nearly all living organisms, including both hosts and invaders. Proteins such as ferritin regulate the iron levels in a cell, and in the event of a pathogenic invasion, the host can use an iron-withholding mechanism to restrict the availability of this essential nutrient to the invading pathogens. However, pathogens use various strategies to overcome this host defense. In this study, we demonstrated that white spot syndrome virus (WSSV) protein kinase 1 (PK1) interacted with shrimp ferritin in the yeast two-hybrid system. A pulldown assay and 27-MHz quartz crystal microbalance (QCM) analysis confirmed the interaction between PK1 and both ferritin and apoferritin. PK1 did not promote the release of iron ions from ferritin, but it prevented apoferritin from binding ferrous ions. When PK1 was overexpressed in Sf9 cells, the cellular labile iron pool (LIP) levels were elevated significantly. Immunoprecipitation and atomic absorption spectrophotometry (AAS) further showed that the number of iron ions bound by ferritin decreased significantly at 24 h post-WSSV infection. Taken together, these results suggest that PK1 prevents apoferritin from iron loading, and thus stabilizes the cellular LIP levels, and that WSSV uses this novel mechanism to counteract the host cell's iron-withholding defense mechanism. IMPORTANCE: We show here that white spot syndrome virus (WSSV) ensures the availability of iron by using a previously unreported mechanism to defeat the host cell's iron-withholding defense mechanism. This defense is often implemented by ferritin, which can bind up to 4,500 iron atoms and acts to sequester free iron within the cell. WSSV's novel counterstrategy is mediated by a direct protein-protein interaction between viral protein kinase 1 (PK1) and host ferritin. PK1 interacts with both ferritin and apoferritin, suppresses apoferritin's ability to sequester free iron ions, and maintains the intracellular labile iron pool (LIP), and thus the availability of free iron is increased within cells.

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.

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).

Characterization and interactome study of white spot syndrome virus envelope protein VP11.

White spot syndrome virus (WSSV) is a large enveloped virus. The WSSV viral particle consists of three structural layers that surround its core DNA: an outer envelope, a tegument and a nucleocapsid. Here we characterize the WSSV structural protein VP11 (WSSV394, GenBank accession number AF440570), and use an interactome approach to analyze the possible associations between this protein and an array of other WSSV and host proteins. Temporal transcription analysis showed that vp11 is an early gene. Western blot hybridization of the intact viral particles and fractionation of the viral components, and immunoelectron microscopy showed that VP11 is an envelope protein. Membrane topology software predicted VP11 to be a type of transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal. Based on an immunofluorescence assay performed on VP11-transfected Sf9 cells and a trypsin digestion analysis of the virion, we conclude that, contrary to topology software prediction, the C-terminal of this protein is in fact inside the virion. Yeast two-hybrid screening combined with co-immunoprecipitation assays found that VP11 directly interacted with at least 12 other WSSV structural proteins as well as itself. An oligomerization assay further showed that VP11 could form dimers. VP11 is also the first reported WSSV structural protein to interact with the major nucleocapsid protein VP664.

The DNA virus white spot syndrome virus uses an internal ribosome entry site for translation of the highly expressed nonstructural protein ICP35.

Although shrimp white spot syndrome virus (WSSV) is a large double-stranded DNA virus (∼300 kbp), it expresses many polycistronic mRNAs that are likely to use internal ribosome entry site (IRES) elements for translation. A polycistronic mRNA encodes the gene of the highly expressed nonstructural protein ICP35, and here we use a dual-luciferase assay to demonstrate that this protein is translated cap independently by an IRES element located in the 5' untranslated region of icp35. A deletion analysis of this region showed that IRES activity was due to stem-loops VII and VIII. A promoterless assay, a reverse transcription-PCR together with quantitative real-time PCR analysis, and a stable stem-loop insertion upstream of the Renilla luciferase open reading frame were used, respectively, to rule out the possibility that cryptic promoter activity, abnormal splicing, or read-through was contributing to the IRES activity. In addition, a Northern blot analysis was used to confirm that only a single bicistronic mRNA was expressed. The importance of ICP35 to viral replication was demonstrated in a double-stranded RNA (dsRNA) interference knockdown experiment in which the mortality of the icp35 dsRNA group was significantly reduced. Tunicamycin was used to show that the α subunit of eukaryotic initiation factor 2 is required for icp35 IRES activity. We also found that the intercalating drug quinacrine significantly inhibited icp35 IRES activity in vitro and reduced the mortality rate and viral copy number in WSSV-challenged shrimp. Lastly, in Sf9 insect cells, we found that knockdown of the gene for the Spodoptera frugiperda 40S ribosomal protein RPS10 decreased icp35 IRES-regulated firefly luciferase activity but had no effect on cap-dependent translation.

Spawning stress triggers WSSV replication in brooders via the activation of shrimp STAT.

In the early days of shrimp aquaculture, wild-captured brooders usually spawned repeatedly once every 2-4days. However, since the first outbreaks of white spot disease (WSD) nearly 20years ago, captured female brooders often died soon after a single spawning. Although these deaths were clearly attributable to WSD, it has always been unclear how spawning stress could lead to an outbreak of the disease. Using real-time qPCR, we show here that while replication of the white spot syndrome virus (WSSV; the causative agent of WSD) is triggered by spawning, there was no such increase in the levels of another shrimp DNA virus, IHHNV (infectious hypodermal and hematopoietic necrosis virus). We also show that levels of activated STAT are increased in brooders during and after spawning, which is important because shrimp STAT is known to transactivate the expression of the WSSV immediate early gene ie1. Lastly, we used dsRNA silencing experiment to show that both WSSV ie1 gene expression and WSSV genome copy number were reduced significantly after shrimp STAT was knocked-down. This is the first report to demonstrate in vivo that shrimp STAT is important for WSSV replication and that spawning stress increases activated STAT, which in turn triggers WSSV replication in WSSV-infected brooders.

Feeding hermit crabs to shrimp broodstock increases their risk of WSSV infection.

White spot syndrome virus (WSSV) is a serious shrimp pathogen that has spread globally to all major shrimp farming areas, causing enormous economic losses. Here we investigate the role of hermit crabs in transmitting WSSV to Penaeus monodon brooders used in hatcheries in Vietnam. WSSV-free brooders became PCR-positive for WSSV within 2 to 14 d, and the source of infection was traced to hermit crabs being used as live feed. Challenging hermit crabs with WSSV confirmed their susceptibility to infection, but they remained tolerant to disease even at virus loads equivalent to those causing acute disease in shrimp. As PCR screening also suggests that WSSV infection occurs commonly in hermit crab populations in both Vietnam and Taiwan, their use as live feed for shrimp brooders is not recommended.

Penaeus monodon thioredoxin restores the DNA binding activity of oxidized white spot syndrome virus IE1.

AIMS: In this study we identified viral gene targets of the important redox regulator thioredoxin (Trx), and explored in depth how Trx interacts with the immediate early gene #1 (IE1) of the white spot syndrome virus (WSSV). RESULTS: In a pull-down assay, we found that recombinant Trx bound to IE1 under oxidizing conditions, and a coimmunoprecipitation assay showed that Trx bound to WSSV IE1 when the transfected cells were subjected to oxidative stress. A pull-down assay with Trx mutants showed that no IE1 binding occurred when cysteine 62 was replaced by serine. Electrophoretic mobility shift assay (EMSA) showed that the DNA binding activity of WSSV IE1 was downregulated under oxidative conditions, and that Penaeus monodon Trx (PmTrx) restored the DNA binding activity of the inactivated, oxidized WSSV IE1. Another EMSA experiment showed that IE1's Cys-X-X-Cys motif and cysteine residue 55 were necessary for DNA binding. Measurement of the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) in WSSV-infected shrimp showed that oxidative stress was significantly increased at 48 h postinfection. The biological significance of Trx was also demonstrated in a double-strand RNA Trx knockdown experiment where suppression of shrimp Trx led to significant decreases in mortality and viral copy numbers. INNOVATION AND CONCLUSION: WSSV's pathogenicity is enhanced by the virus' use of host Trx to rescue the DNA binding activity of WSSV IE1 under oxidizing conditions.

Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon.

BACKGROUND: The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the P. monodon genome were obtained for repetitive and protein-coding sequence analyses. RESULTS: We found that microsatellite sequences were highly abundant in the P. monodon genome, comprising 8.3% of the total length. The density and the average length of microsatellites were evidently higher in comparison to those of other taxa. AT-rich microsatellite motifs, especially poly (AT) and poly (AAT), were the most abundant. High abundance of microsatellite sequences were also found in the transcribed regions. Furthermore, via self-BlastN analysis we identified 103 novel repetitive element families which were categorized into four groups, i.e., 33 WSSV-like repeats, 14 retrotransposons, 5 gene-like repeats, and 51 unannotated repeats. Overall, various types of repeats comprise 51.18% of the P. monodon genome in length. Approximately 7.4% of the FESs contained protein-coding sequences, and the Inhibitor of Apoptosis Protein (IAP) gene and the Innexin 3 gene homologues appear to be present in high abundance in the P. monodon genome. CONCLUSIONS: The redundancy of various repeat types in the P. monodon genome illustrates its highly repetitive nature. In particular, long and dense microsatellite sequences as well as abundant WSSV-like sequences highlight the uniqueness of genome organization of penaeid shrimp from those of other taxa. These results provide substantial improvement to our current knowledge not only for shrimp but also for marine crustaceans of large genome size.

Penaeus monodon TATA box-binding protein interacts with the white spot syndrome virus transactivator IE1 and promotes its transcriptional activity.

We show here that the white spot syndrome virus (WSSV) immediate-early protein IE1 interacts with the Penaeus monodon TATA box-binding protein (PmTBP) and that this protein-protein interaction occurs in the absence of any other viral or cellular proteins or nucleic acids, both in vitro and in vivo. Mapping studies using enhanced green fluorescent protein (EGFP) fusion proteins containing truncations of IE1 and PmTBP delimited the interacting regions to amino acids (aa) 81 to 180 in IE1 and, except for aa 171 to 230, to aa 111 to 300 in PmTBP. A WSSV IE1 transactivation assay showed that large quantities (>800 ng) of the GAL4-IE1 plasmid caused "squelching" of the GAL4-IE1 activity and that this squelching effect was alleviated by the overexpression of PmTBP. Gene silencing of WSSV ie1 and PmTBP by pretreatment with double-stranded RNAs (dsRNAs) prior to WSSV challenge showed that the expression of these two target genes was specifically inhibited by their corresponding dsRNAs 72 and 96 h after dsRNA treatment. dsRNA silencing of ie1 and PmTBP expression also significantly reduced WSSV replication and the expression of the viral early gene dnapol (DNA polymerase gene). These results suggest that WSSV IE1 and PmTBP work cooperatively with each other during transcription initiation and, furthermore, that PmTBP is an important target for WSSV IE1's transactivation activity that can enhance viral gene expression and help in virus replication.

Assessment of the roles of copepod Apocyclops royi and bivalve mollusk Meretrix lusoria in white spot syndrome virus transmission.

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.

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.

Molecular mechanism of the interactions between white spot syndrome virus anti-apoptosis protein AAP-1 (WSSV449) and shrimp effector caspase.

AAP-1 (WSSV449), an anti-apoptosis protein encoded by white spot syndrome virus (WSSV), blocked apoptosis in insect cells (SF9) induced by Penaeus monodon effector caspase (Pm caspase). Here, to characterize in detail the anti-Pm caspase activity of AAP-1, both proteins were expressed and purified from Escherichia coli and their interactions were assayed in vitro. We found that although AAP-1 could inhibit Pm caspase activity, the inhibition was not as efficient as that of baculovirus anti-apoptosis protein P35. We further confirmed the binding and cleavage of AAP-1 by Pm caspase, and detected three AAP-1 cleavage products. Mutational analysis and protein N-terminal sequencing revealed that whereas both Asp233 and Asp272 residues of AAP-1 are involved in binding and cleavage by Pm caspase, only the Asp272 is involved in Pm caspase inhibition. Asp233, on the other hand, negatively regulates AAP-1's anti-Pm caspase activity. Lastly, AAP-1 homotypically interacts with each other both in vitro and in insect cells.

A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins.

BACKGROUND: Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus), is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process. PRINCIPAL FINDINGS: In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument) proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers. CONCLUSIONS: From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented.

Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages.

Complementary (c)DNA encoding glutathione peroxidase (GPx) messenger (m)RNA of the tiger shrimp Penaeus monodon was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. The 1321-bp cDNA contained an open reading frame (ORF) of 564bp, a 69-bp 5'-untranslated region (UTR), and a 688-bp 3'-UTR containing a poly A tail and a conserved selenocysteine insertion sequence (SECIS) element. The molecular mass of the deduced amino acid (aa) sequence (188 aa) was 21.05kDa long with an estimated pI of 7.68. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, (190)TGA(192), and forms the active site with residues Glu(75) and Trp(143). Comparison of amino acid sequences showed that tiger shrimp GPx is more closely related to vertebrate GPx1, in accordance with those in Litopenaeus vannamei and Macrobrachium rosenbergii. GPx cDNA was synthesised in lymphoid organ, gills, heart, haemocytes, the hepatopancreas, muscles, and intestines. After injected with either Photobacterium damsela or white spot syndrome virus (WSSV), the respiratory bursts of shrimp significantly increased in order to kill the pathogen, and induced increases in the activities of superoxide dismutase and GPx, and regulation in the expression of cloned GPx mRNA to protect cells against damage from oxidation. The GPx expression significantly increased at stage D(0/1), and then gradually decreased until stage C suggesting that the cloned GPx might play a role in the molt regulation of shrimp.

Polycistronic mRNAs and internal ribosome entry site elements (IRES) are widely used by white spot syndrome virus (WSSV) structural protein genes.

The genome of the white spot syndrome virus (WSSV) Taiwan isolate has many structural and non-structural genes that are arranged in clusters. Screening with Northern blots showed that at least four of these clusters produce polycistronic mRNA, and one of these (vp31/vp39b/vp11) was studied in detail. The vp31/vp39b/vp11 cluster produces two transcripts, including a large 3.4-kb polycistronic transcript of all three genes. No monocistronic vp39b mRNA was detected. TNT and in vitro translation assays showed that vp39b translation was independent of vp31 translation, and that ribosomal reinitiation was not a possible mechanism for vp39b. An unusually located IRES (internal ribosome entry site) element was identified in the vp31/vp39b coding region, and this region was able to promote the expression of a downstream firefly luciferase reporter. We show that vp31/vp39b/vp11 is representative of many other WSSV structural/non-structural gene clusters, and argue that these are also likely to produce polycistronic mRNAs and that use an IRES mechanism to regulate their translation.

Characterization of white spot syndrome virus envelope protein VP51A and its interaction with viral tegument protein VP26.

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.