White Spot Syndrome Virus Envelope Protein Research Articles

A computational approach to identifying peptide inhibitors againstWhite Spot Syndrome Virus: Targeting the virus envelope protein

The white spot syndrome virus (WSSV), a rapidly replicating and highly lethal pathogen that targets Penaeid shrimp, has emerged as one of the most widespread viruses globally due to its high virulence. With effective chemotherapeutics still unavailable, the pursuit of novel and viable strategies against WSSV remains a crucial focus in the field of shrimp farming. The envelope proteins of WSSV are essential for virus entry, serving as excellent targets for the development of antiviral therapeutics. Novel strategies in the design of inhibitory peptides, especially those targeting envelope protein (VP28) located on the surface of the virus particle, play a critical role as a significant virulence factor during the early stages of inherent WSSV infection in shrimp. In this direction, the current computational study focused on identifying self-inhibitory peptides from the hydrophobic membrane regions of the VP28 protein, employing peptide docking and molecular dynamics simulation (MDS) approaches. Such inhibitory peptides could be useful building blocks for the rational engineering of inhibitory therapeutics since they imitate the mechanism of binding to homologous partners used by their origin domain to interact with other molecules. The N-terminal sequence of VP28 has been reported as the potential site for membrane interactions during the virus entry. Moreover, drug delivery systems mediated by chitosan and gold nanoparticles are being developed to enhance the therapeutic efficacy of anti-viral peptides. These systems can increase the solubility, stability, and selectivity of peptides, possessing better qualities than conventional delivery methods. This computational study on self-inhibitory peptides could be a valuable resource for further in vitro and in vivo studies on anti-viral therapeutics in the aquaculture industry.

White spot syndrome virus hijacks host PP2A-FOXO axes to promote its propagation

Viruses have developed superior strategies to escape host defenses or exploit host components and enable their infection. The forkhead box transcription factor O family proteins (FOXOs) are reportedly utilized by human cytomegalovirus during their reactivation in mammals, but if FOXOs are exploited by viruses during their infection remains unclear. In the present study, we found that the FOXO of kuruma shrimp (Marsupenaeus japonicus) was hijacked by white spot syndrome virus (WSSV) during infection. Mechanistically, the expression of leucine carboxyl methyl transferase 1 (LCMT1) was up-regulated during the early stages of WSSV infection, which activated the protein phosphatase 2A (PP2A) by methylation, leading to dephosphorylation of FOXO and translocation into the nucleus. The FOXO directly promoted transcription of the immediate early gene, wsv079 of WSSV, which functioned as a transcriptional activator to initiate the expression of viral early and late genes. Thus, WSSV utilized the host LCMT1-PP2A-FOXO axis to promote its replication during the early infection stage. We also found that, during the late stages of WSSV infection, the envelope protein of WSSV (VP26) promoted PP2A activity by directly binding to FOXO and the regulatory subunit of PP2A (B55), which further facilitated FOXO dephosphorylation and WSSV replication via the VP26-PP2A-FOXO axis in shrimp. Overall, this study reveals novel viral strategies by which WSSV hijacks host LCMT1-PP2A-FOXO or VP26-PP2A-FOXO axes to promote its propagation, and provides clinical targets for WSSV control in shrimp aquaculture.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential “nitrite stress–Duox activation–ROS production” pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for “nitrite stress–Duox activation–ROS production–Dorsal activation–AMP synthesis” that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

A Novel Hemocyte-Specific Small Protein Participates in White Spot Syndrome Virus Infection via Binding to Viral Envelope Protein.

Hemocytes are essential components of the immune system against invading pathogens in shrimp. Many uncharacterized transcripts exist in hemocytes but the knowledge of them is very limited. In the present study, we identified a novel small protein from the uncharacterized transcripts in hemocytes of Litopenaeus vannamei. This transcript was specifically expressed in hemocytes and encoded a novel secretory protein, which was designated as hemocyte-specific small protein (LvHSSP). The expression level of LvHSSP was significantly up-regulated in the hemocytes of shrimp infected with white spot syndrome virus (WSSV). After knockdown of LvHSSP by RNA interference, the WSSV copy number in shrimp decreased significantly. Conversely, WSSV copy number increased in shrimp when they were infected by WSSV after incubation with recombinant LvHSSP protein. These results suggested that LvHSSP might promote viral infection in shrimp. Immunocytochemical assay showed that the recombinant LvHSSP protein was located on the membrane of hemocytes. Co-IP results showed that LvHSSP could interact with VP26, the main envelope protein of WSSV, suggesting that LvHSSP might mediate WSSV adhesion and entry into host cells by binding to viral envelope protein. Meanwhile, the total hemocyte counts were significantly decreased after LvHSSP knockdown while increased after supplementing with recombinant LvHSSP protein, supporting the idea of hemocytes as the carrier for systemic dissemination of WSSV. This study reported a novel small protein in hemocytes, which modulated the viral infection in shrimp. Our results will enrich the knowledge of invertebrate innate immunity and provide a new field in the study of hemocyte function.

Study of effects of dietary quercetin (Que) on growth performance and disease resistance mechanism of Litopenaeus vannamei

The purpose of this study was to evaluate effects of dietary quercetin (Que) on growth performance and disease resistance mechanism of Litopenaeus vannamei. Shrimp were randomly divided into 4 groups, with 3 replicants in each group and 70 shrimp in each replicants. The contents of Que. in the four groups were 0, 0.25, 0.5 and 1 g kg−1, respectively. Samples were taken after 56 days, followed by a 3-day white spot syndrome virus (WSSV) challenge experiment. The results showed that Que. significantly improved the growth performance and reduced the content of crude fat in shrimp (P < 0.05). Dietary Que. increased phagocytic activity, bacteriolytic activity and antibacterial activity (P < 0.05), but had no effects on the total hemocytes count. Dietary Que. also improved the expression levels of genes related to immunity of shrimp. In addition, dietary Que. improved the activities of catalase and glutathione peroxidase, total antioxidant capability and the content of glutathione (P < 0.05), but decreased the superoxide dismutase activity (P < 0.05). In the case of intestinal microbiota, Que. reduced microbial diversity and richness. Meanwhile, Que. also increased abundances of functional genes related to the “immune system” and “lipid metabolism”. After WSSV challenge, the cumulative mortality of shrimp decreased with increasing levels of dietary Que., the gene expression of WSSV envelope protein and hemocytes surface receptor protein were down-regulated (P < 0.05), while the gene expression of hemocytes immunity was up-regulated (P < 0.05). In summary, Que. improved the growth performance, enhanced the antioxidant capability and immune response, regulated the composition and structure of intestinal microbiota, and reduced cumulative mortality after WSSV challenge of Litopenaeus vannamei.

Lysyl Oxidase-like Protein Recognizes Viral Envelope Proteins and Bacterial Polysaccharides against Pathogen Infection via Induction of Expression of Antimicrobial Peptides

Lysyl oxidases (LOXs) are copper-dependent monoamine oxidases, and they play critical roles in extracellular matrix (ECM) remodeling. The LOX and LOX-like (LOXL) proteins also have a variety of biological functions, such as development and growth regulation, tumor suppression, and cellular senescence. However, the functions of LOXLs containing repeated scavenger receptor cysteine-rich (SRCR) domains in immunity are rarely reported. In this study, we characterized the antiviral and antibacterial functions of a lysyl oxidase-like (LOXL) protein containing tandem SRCR domains in Marsupenaeus japonicus. The mRNA level of LoxL was significantly upregulated in the hemocytes and intestines of shrimp challenged using white spot syndrome virus (WSSV) or bacteria. After the knockdown of LoxL via RNA interference, WSSV replication and bacterial loads were apparently increased, and the survival rate of the shrimp decreased significantly, suggesting that LOXL functions against pathogen infection in shrimp. Mechanistically, LOXL interacted with the envelope proteins of WSSV or with lipopolysaccharide and peptidoglycan from bacteria in shrimp challenged using WSSV or bacteria, and it promoted the expression of a battery of antimicrobial peptides (AMPs) via the induction of Dorsal nuclear translocation against viral and bacterial infection. Moreover, LOXL expression was also positively regulated by Dorsal in the shrimp challenged by pathogens. These results indicate that, by acting as a pattern recognition receptor, LOXL plays vital roles in antiviral and antibacterial innate immunity by enhancing the expression of AMPs in shrimp.

Identification of three novel Spätzle genes in Eriocheir sinensis and their roles during white spot syndrome virus infection

Proteins of Spätzle family play an essential role in innate immunity in invertebrates by activating the Toll pathway to induce the expression of antimicrobial peptides. However, little is known about the function of Spätzle in in the immune response of the Chinese mitten crab. In the present study, three novel Spätzle genes (named as EsSpz1, EsSpz2, and EsSpz3) were identified from Eriocheir sinensis. The genome structure of EsSpz1 contains two exons and an intron. Three Spätzle proteins all contain a Pfam Spaetzle domain. In the evolution, EsSpz1-3 cluster with other Spätzle proteins from crustaceans. EsSpz1-3 were widely distributed in multiple immune tissues. The expression levels of EsSpz1-3 in the intestine were remarkably upregulated after white spot syndrome virus (WSSV) challenge. The knockdown of EsSpz1-3 remarkably decreased the expressions of crustins and anti-lipopolysaccharide factors during WSSV infection. Moreover, EsSpz1-3 silencing remarkably increased the expression of WSSV envelope protein VP28. These findings suggest that new-found EsSpz1-3 in E. sinensis could promote the synthesis of antimicrobial peptides and inhibit the expression of VP28 during WSSV infection. Our study indicates that EsSpz1-3 in E. sinensis may participate in the innate immune defenses against WSSV by inducing the expression of antimicrobial peptides. This study provides new knowledge for the function of Spätzle in the antiviral immune defense in crustacean.

Characterization of four spliced isoforms of a transmembrane C-type lectin from Procambarus clarkii and their function in facilitating WSSV infection

C-type lectin (CTL) is an important pattern recognition receptor that play vital functions in the innate immunity. Many soluble CTLs in crustacean participate in the inhibition or promotion of white spot syndrome virus (WSSV) infection. However, whether transmembrane CTLs participate in WSSV infection in crustacean remains unknown. In the present study, four spliced isoforms of a transmembrane CTL (designated as PcTlec) from Procambarus clarkii were identified for the first time. The genome structure of PcTlec contains eight exons, six known introns, and one unknown intron. PcTlec-isoform1 is produced by intron retention, whereas PcTlec-isoform3 and PcTlec-isoform4 are produced by exon skipping. All of them contain the transmembrane domain and characteristic carbohydrate recognition domain (CRD). Four PcTlec isoforms were mainly expressed in the hepatopancreas, stomach, and intestine. After WSSV challenge, the expression levels of PcTlec-isoform1-4 in the intestine were upregulated. The knockdown of the region shared by four PcTlec isoforms evidently decreased the expression of WSSV envelope protein VP28 and the copies of viral particles. A recombinant protein (rPcTlec-CRD) containing the CRD that was shared by four PcTlec isoforms was acquired by procaryotic expression system. The injection of purified rPcTlec-CRD protein evidently increased the VP28 expression and WSSV copies during viral infection. Moreover, rPcTlec-CRD could directly bind to WSSV and interact with VP28 protein. These findings indicate that new-found transmembrane CTL isoforms in P. clarkii may act as viral receptors that facilitate WSSV infection. This study contributes to the recognition and understanding of the functions of transmembrane CTLs in crustacean in the infection of host by WSSV.

Applying Modified VP53A Recombinant Protein as an Anti-White Spot Syndrome Virus Biological Agent in Litopenaeus vannamei Farming.

Shrimp farming is an important economic activity. However, due to the spread of pathogens, shrimp aquaculture is becoming increasingly difficult. Many studies have confirmed that white spot syndrome virus (WSSV) recombinant proteins can inhibit viral infection. Among them, VP53 recombinant protein has been found to reduce mortality upon WSSV challenge. This study was conducted in Kaohsiung, Taiwan and reports the first field feeding trial to demonstrate that WSSV recombinant proteins can improve shrimp survival rates at a farming scale. Prior to the feeding trial, the shrimp were confirmed to be slightly infected with WSSV, Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND), non-AHPND V. parahaemolyticus strains, and Enterocytozoon hepatopenaei (EHP), which are common pathogens that shrimp farmers often face. The shrimp were then divided into two groups: a control group (C group) fed with a commercial diet and a protein group (P group) fed with the same commercial feed with VP53 recombinant protein. Our findings indicated that the survival rate and expression of immune genes of the P group were higher than those of the C group. The intestinal microbiota of the two groups were also analysed. Collectively, our results confirmed that the recombinant WSSV envelope protein derivative can be used as an effective anti-virus biological agent in shrimp farms.

The dyslexia-associated KIAA0319-like protein from Marsupenaeus japonicus recognizes white spot syndrome virus and restricts its replication

Dyslexia-associated protein KIAA0319, with immunoglobulin-like polycystic kidney disease (PKD) domains, is a dyslexia susceptibility protein. KIAA0319 participates in neuronal migration and is involved in the interaction between neurons and glial fibers. Recent studies found that KIAA0319 acts as a viral receptor via an interaction between its PKD domains with viruses. KIAA0319 mediates viral internalization via a clathrin-dependent pathway involving interaction with adaptor protein 2 (AP2). In the present study, we identified a cDNA encoding a dyslexia-associated KIAA0319-like protein from Marsupenaeus japonicus (designated as MjKIAA0319L). MjKIAA0319L was upregulated in shrimp challenged by white spot syndrome virus (WSSV). After knockdown of MjKIAA0319L using RNA interference, followed by WSSV infection in shrimp, WSSV replication increased and the shrimp survival rate decreased significantly. Further study found that the extracellular domain of MjKIAA0319L expressed in Escherichia coli bound WSSV particles by interacting with the WSSV envelope protein VP28. Fluorescent immunocytochemical analysis showed that MjKIAA0319L was internalized into the cytoplasm of hemocytes during WSSV infection. The intracellular domain of MjKIAA0319L, comprising a YXXΦ motif, interacted with AP2μ, suggesting that MjKIAA0319L binds to WSSV and induces endocytosis in a clathrin-dependent manner. Therefore, MjKIAA0319L recognizes WSSV via its extracellular domain and induces endocytosis of WSSV through its intracellular domain in shrimp. Thus, the MjKIAA0319L-AP2-clathrin pathway is involved in the phagocytosis of WSSV to restrict viral infection in shrimp.

SpSR-B2 functions as a potential pattern recognition receptor involved in antiviral and antibacterial immune responses of mud crab Scylla paramamosain

Although class B scavenger receptors (SR-Bs) in mammals are multifunctional molecules, the functions of SR-Bs in invertebrates remain largely unknown. In this study, we characterized an SR-B homolog, namely SpSR-B2, from Scylla paramamosain. SpSR-B2 shared high similarity with mammalian SR-Bs, and exhibited specific binding activity to ac-LDL, indicating that it may be a new member of SR-B class in invertebrates. SpSR-B2 was upregulated after challenge with white spot syndrome virus (WSSV) or bacteria. Binding assays showed that SpSR-B2 specifically interacted with WSSV envelope protein VP24. Besides, SpSR-B2 could bind to all tested bacterial cells and agglutinate these bacteria. SpSR-B2 also exhibited a strong binding activity to LPS but weak binding activities to other tested polysaccharides. These findings indicated that SpSR-B2 was a potential recognition molecule for viral protein VP24 and bacterial LPS. Knockdown of SpSR-B2 resulted in dramatically decreased expressions of certain antimicrobial peptides (AMPs), and overexpression of SpSR-B2 led to the increased expression of the AMP of SpALF2, suggesting that SpSR-B2 could regulate the expression of AMPs. Taken together, this study revealed that SpSR-B2 functioned as a potential pattern recognition receptor participating in antiviral and antibacterial immunity, and provided new insights into the immune functions of invertebrate SR-Bs.

Anti-lipopolysaccharide factor D from kuruma shrimp exhibits antiviral activity

Anti-lipopolysaccharide factors (ALFs) exhibit a potent antimicrobial activity against a broad range of bacteria, filamentous fungi, and viruses. In previous reports, seven groups of ALFs (groups A–G) were identified in penaeid shrimp. Among them, group D showed negative net charges and weak antimicrobial activity. Whether this group has antiviral function is not clear. In this study, the ALF sequences of penaeid shrimp were analyzed, and eight groups of ALF family (groups A–H) were identified. The four ALFs including MjALF-C2, MjALF-D1, MjALF-D2, and MjALF-E2 from kuruma shrimp Marsupenaeus japonicus were expressed recombinantly in Escherichia coli, and the antiviral activity was screened via injection of purified recombinant ALFs into shrimp following white spot syndrome virus (WSSV) infection. Results showed that the expression of Vp28 (WSSV envelope protein) decreased significantly in the MjALF-D2-injected shrimp only. Therefore, MjALF-D2 was chosen for further study. Expression pattern analysis showed that MjAlf-D2 was upregulated in shrimp challenged by WSSV. The WSSV replication was detected in RNA, genomic DNA, and protein levels using VP28 and Ie1 (immediate-early gene of WSSV) as indicators in MjALF-D2-injected shrimp following WSSV infection. Results showed that WSSV replication was significantly inhibited compared with that in the rTRX- or PBS-injected control groups. After knockdown of MjAlf-D2 in shrimp by RNA interference, the WSSV replication increased significantly in the shrimp. All these results suggested that MjALF-D2 has an antiviral function in shrimp immunity, and the recombinant ALF-D2 has a potential application for viral disease control in shrimp aquaculture.

Identification of a Shrimp E3 Ubiquitin Ligase TRIM50-Like Involved in Restricting White Spot Syndrome Virus Proliferation by Its Mediated Autophagy and Ubiquitination.

Most tripartite motif (TRIM) family proteins are critical components of the autophagy machinery and play important roles in host defense against viral pathogens in mammals. However, the roles of TRIM proteins in autophagy and viral infection have not been studied in lower invertebrates, especially crustaceans. In this study, we first identified a TRIM50-like gene from Penaeus monodon (designated PmTRIM50-like), which, after a white spot syndrome virus (WSSV) challenge, was significantly upregulated at the mRNA and protein levels in the intestine and hemocytes. Knockdown of PmTRIM50-like led to an increase in the WSSV quantity in shrimp, while its overexpression led to a decrease compared with the controls. Autophagy can be induced by WSSV or rapamycin challenge and has been shown to play a positive role in restricting WSSV replication in P. monodon. The mRNA and protein expression levels of PmTRIM50-like significantly increased with the enhancement of rapamycin-induced autophagy. The autophagy activity induced by WSSV or rapamycin challenge could be inhibited by silencing PmTRIM50-like in shrimp. Further studies showed that rapamycin failed to induce autophagy or inhibit WSSV replication after knockdown of PmTRIM50-like. Moreover, pull-down and in vitro ubiquitination assays demonstrated that PmTRIM50-like could interact with WSSV envelope proteins and target them for ubiquitination in vitro. Collectively, this study demonstrated that PmTRIM50-like is required for autophagy and is involved in restricting the proliferation of WSSV through its ubiquitination. This is the first study to report the role of a TRIM family protein in virus infection and host autophagy in crustaceans.

Development of a VP28-pseudotyped baculovirus expression system for efficient gene transfer in penaeid shrimps

Both efficient gene transfer and expression tools and suitable reporter genes are lacking for penaeid shrimps and their isolated cells. In this study a VP28-pseudotyped insect baculovirus expression system: Bacmid-GUS/VP28, carrying a reporter gene of GUS (β-glucuronidase) and an exogenous envelope protein of VP28 from shrimp white spot syndrome virus (WSSV), has been successfully developed for the first time and well used for efficient gene transfer and expression in adult shrimps with an extremely high infection efficiency of up to 100%, but in a tissue-specific manner because it could only infect the adult tissues of gill, heart and intestine, but not the Oka organ and muscle under the tested infection doses. The aforementioned VP28-pseudotyped recombinant baculovirus of Bacmid-GUS/VP28 could also infect the primarily cultured hemolymph cells of shrimp (Metapenaeus ensiss) and produced strong GUS signals with a relatively low efficiency of 1.21%, but failed in the primary embryonic cells of shrimp (Penaeus japonicus), showing an obvious cell-specificity. A possible explanation for the above-mentioned low infection efficiency is the occurrence of mitosis-arrest in the infected shrimp cells. In contrast, no obvious GUS signals could be detected in any of the uninfected and wild type recombinant baculovirus (Bacmid-GUS)-infected tissues and cells of adult shrimps, confirming the suitability of GUS as a reporter gene and the great contribution of VP28 to the tissue- and cell- tropism of pseudotyped recombinant baculovirus (Bacmid-GUS/VP28) in penaeid shrimps. In a word, the successful development of VP28-pseudotyped recombinant baculovirus system will provide us a useful tool for efficient gene transfer and expression in adult shrimps as well as an assay system for the examination of the contribution of different WSSV envelope proteins to the viral tissue tropism.

Prediction of Interaction Sites of PcRab7-VP28

White spot syndrome virus (WSSV) is one of the most important pathogens in the world. Since its outbreak in 1993, the virus has caused huge economic losses. Studies have confirmed that in the early stage of infection, VP28, the main envelope protein of WSSV, as a viral adhesion protein, binds to PcRab7 of Penaeus chinensis to help the virus enter the host cells. Understanding the mechanism of PcRab7-VP28 interaction is of great significance to understand the mechanism of WSSV infection and the development of antiviral drugs. In this research, the interaction interface and interaction sites were predicted by using the methods of molecular simulations. Results showed that VP28 binds to the second β-sheet (L73-D86) of PcRab7, which is consistent with the region detected in previous studies. Furthermore, we speculated the possible interaction sites in PcRab7 are E81, F77 and D76. These results may contribute to a deep understanding of the infection mechanism of WSSV on the host.

Identification and functional characterization of a novel C-type lectin from the kuruma shrimp, Marsupenaeus japonicus

C-type lectins (CTLs) are immune molecules that are crucial to the invertebrate innate immune system with the primary function of recognizing invading pathogens. In the present study, a novel CTL was cloned from Marsupenaeus japonicus (MjCTL), and its tissue distribution and expression patterns over time in response to white spot syndrome virus (WSSV) and Vibrio parahaemolyticus were further investigated. The open reading frame (ORF) of MjCTL was 513 bp and encoded a polypeptide of 170 amino acids, which contained a signal peptide and a carbohydrate recognition domain (CRD) that are typical for CTLs. MjCTL was primarily expressed in the hepatopancreas and weakly expressed in hemocytes, gill, stomach, intestine, heart, muscle and eyestalk. The expression level of MjCTL in the hepatopancreas was dramatically increased at 48 h post-injection with WSSV at a dosage of 1 × 105 virions. Glutathione-S-transferase (GST) pull-down assays showed that MjCTL could directly bind to several WSSV envelope proteins, including VP19, VP24, VP26 and VP28. Moreover, MjCTL displayed antibacterial activity against V. parahaemolyticus. Our results indicated that MjCTL exhibited multiple functions in innate immune response against pathogens.

A shrimp glycosylase protein, PmENGase, interacts with WSSV envelope protein VP41B and is involved in WSSV pathogenesis

Viral glycoproteins are expressed by many viruses, and during infection they usually play very important roles, such as receptor attachment or membrane fusion. The mature virion of the white spot syndrome virus (WSSV) is unusual in that it contains no glycosylated proteins, and there are currently no reports of any glycosylation mechanisms in the pathogenesis of this virus. In this study, we cloned a glycosylase, mannosyl-glycoprotein endo-β-N-acetylglucosaminidase (ENGase, EC 3.2.1.96), from Penaeus monodon and found that it was significantly up-regulated in WSSV-infected shrimp. A yeast two-hybrid assay showed that PmENGase interacted with both structural and non-structural proteins, and GST-pull down and co-immunoprecipitation (Co-IP) assays confirmed its interaction with the envelope protein VP41B. In the WSSV challenge tests, the cumulative mortality and viral copy number were significantly decreased in the PmEngase-silenced shrimp, from which we conclude that shrimp glycosylase interacts with WSSV in a way that benefits the virus. Lastly, we speculate that the deglycosylation activity of PmENGase might account for the absence of glycosylated proteins in the WSSV virion.

Identification and Molecular Characterization of a Pellino Protein in Kuruma Prawn (Marsupenaeus Japonicus) in Response to White Spot Syndrome Virus and Vibrio Parahaemolyticus Infection.

Kuruma prawn, Marsupenaeus japonicus, has the third largest annual yield among shrimp species with vital economic significance in China. White spot syndrome virus (WSSV) is a great threat to the global shrimp farming industry and results in high mortality. Pellino, a highly conserved E3 ubiquitin ligase, has been found to be an important modulator of the Toll-like receptor (TLR) signaling pathways that participate in the innate immune response and ubiquitination. In the present study, the Pellino gene from Marsupenaeus japonicus was identified. A qRT-PCR assay showed the presence of MjPellino in all the tested tissues and revealed that the transcript level of this gene was significantly upregulated in both the gills and hemocytes after challenge with WSSV and Vibrio parahaemolyticus. The function of MjPellino was further verified at the protein level. The results of the three-dimensional modeling and protein–protein docking analyses and a GST pull-down assay revealed that the MjPellino protein was able to bind to the WSSV envelope protein VP26. In addition, the knockdown of MjPellino in vivo significantly decreased the expression of MjAMPs. These results suggest that MjPellino might play an important role in the immune response of kuruma prawn.

Penaeidins restrict white spot syndrome virus infection by antagonizing the envelope proteins to block viral entry

ABSTRACT Emerging studies have indicated that some penaeidins restrict virus infection; however, the mechanism(s) involved are poorly understood. In the present study, we uncovered that penaeidins are a novel family of antiviral effectors against white spot syndrome virus (WSSV), which antagonize the envelope proteins to block viral entry. We found that the expression levels of four identified penaeidins from Litopenaeus vannamei, including BigPEN, PEN2, PEN3, and PEN4, were significantly induced in hemocytes during the early stage of WSSV infection. Knockdown of each penaeidin in vivo via RNA interference resulted in elevated viral loads and rendered shrimp more susceptible to WSSV, while the survival rate was rescued via the injection of recombinant penaeidins. All penaeidins, except PEN4, were shown to interact with several envelope proteins of WSSV, and all four penaeidins were observed to be located on the outer surface of the WSSV virion. Co-incubation of each recombinant penaeidin with WSSV inhibited virion internalization into hemocytes. More importantly, we found that PEN2 competitively bound to the envelope protein VP24 to release it from polymeric immunoglobulin receptor (pIgR), the cellular receptor required for WSSV infection. Moreover, we also demonstrated that BigPEN was able to bind to VP28 of WSSV, which disrupted the interaction between VP28 and Rab7 – the Rab GTPase that contributes to viral entry by binding with VP28. Taken together, our results demonstrated that penaeidins interact with the envelope proteins of WSSV to block multiple viral infection processes, thereby protecting the host against WSSV.

RPS27, a sORF-Encoded Polypeptide, Functions Antivirally by Activating the NF-κB Pathway and Interacting With Viral Envelope Proteins in Shrimp.

A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of <100 amino acids in eukaryotes (50 amino acids in prokaryotes). Studies have shown that several sORF-encoded peptides (SEPs) have important physiological functions in different organisms. Many ribosomal proteins belonging to SEPs play important roles in several cellular processes, such as DNA damage repair and apoptosis. Several studies have implicated SEPs in response to infection and innate immunity, but the mechanisms have been unclear for most of them. In this study, we identified a sORF-encoded ribosomal protein S27 (RPS27) in Marsupenaeus japonicus. The expression of MjRPS27 was significantly upregulated in shrimp infected with white spot syndrome virus (WSSV). After knockdown of MjRPS27 by RNA interference, WSSV replication increased significantly. Conversely, after MjRPS27 overexpression, WSSV replication decreased in shrimp and the survival rate of the shrimp increased significantly. These results suggested that MjRPS27 inhibited viral replication. Further study showed that, after MjRPS27 knockdown, the mRNA expression level of MjDorsal, MjRelish, and antimicrobial peptides (AMPs) decreased, and the nuclear translocation of MjDorsal and MjRelish into the nucleus also decreased. These findings indicated that MjRPS27 might activate the NF-κB pathway and regulate the expression of AMPs in shrimp after WSSV challenge, thereby inhibiting viral replication. We also found that MjRPS27 interacted with WSSV's envelope proteins, including VP19, VP24, and VP28, suggesting that MjRPS27 may inhibit WSSV proliferation by preventing virion assembly in shrimp. This study was the first to elucidate the function of the ribosomal protein MjRPS27 in the antiviral immunity of shrimp.