The C-terminal region of envelope protein VP38 from white spot syndrome virus is indispensable for interaction with VP24

White spot syndrome virus (WSSV) is a large, rod-shaped, enveloped double-stranded DNA virus. In this study, VP38, a viral envelope protein, was expressed as a glutathione S-transferase (GST) fusion protein, and a polyclonal antibody against VP38 was obtained. Far-Western blotting and GST pull-down showed that VP38 interacted directly with VP24, a major WSSV envelope protein. In addition, to delineate the interaction region of VP38 with VP24, GST-VP38n (aa 1–142) and GST-VP38c (aa 143–309) were expressed. The GST pull-down assay revealed that VP38 binds via its C-terminal region to VP24. The result implies that VP38 may participate in the formation of the WSSV envelope.     

Analysis of white spot syndrome virus envelope protein complexome by two-dimensional blue native/SDS PAGE combined with mass spectrometry

White spot syndrome virus (WSSV) is a large enveloped virus, but the organization of its envelope proteins remains largely unknown. In the present study, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and SDS-PAGE in combination with mass spectrometry to analyze the envelope protein complexome of WSSV. Our results show that the viral envelope consists of multi-protein complexes (MPCs). Within them, the envelope protein VP19 exists as a homotrimer, while another major envelope protein, VP28, mainly exists as a homotetramer. The most notable feature is that the majority of MPCs include VP26 and VP24, suggesting that these two proteins might serve as hub proteins to recruit low-abundance proteins to MPCs and play crucial roles in the process of protein complex formation. Furthermore, we found significant evidence for interactions between several low-abundance proteins, such as VP52B/VP38/VP33 and VP12/VP150. The result of this study may promote the further research on WSSV envelope assembly.     

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS–PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.     

The RGD motif in VP31 of white spot syndrome virus is involved in cell adhesion

Two WSSV envelope proteins, VP31 and VP33, contain a conserved Arg-Gly-Asp (RGD) sequence. In order to investigate the role of the RGD motif, wild-type and RGD-mutated VP31 and VP33 were recombinantly expressed in E. coli. The cell adhesion ability of the proteins was investigated in crayfish haemocytes using a fluorescence assay. The results showed that recombinant wild-type VP31 and VP33 had cell adhesion activity, and the RGD motif in VP31 was required for cell adhesion, which could be inhibited by an RGDT peptide. In contrast, the interaction of VP33 with cells did not require the RGD motif. These data indicate that the RGD motif plays an important role in the interaction between VP31 and host cells.     

Interaction of white spot syndrome virus VP26 protein with actin

VP26 protein, the product of the WSV311 gene of white spot syndrome virus (WSSV), is one of major structural proteins of virus. In this study, when purified virions were treated with Triton X-100 detergent, VP26 protein was present in both the envelope and the nucleocapsid fraction. We have rationalized this finding by suggesting that VP26 protein might be located in the space between the envelope and the nucleocapsid. By using a fluorescent probe method, we have investigated the interaction between VP26 protein and some proteins of host cells. Three major VP26-binding proteins were purified from crayfish hemocytes by affinity-chromatography, in which the protein with an apparent molecular mass of 42 kDa was identified as actin by mass spectrometry (MS). Moreover, the association of VP26 protein with actin microfilaments was confirmed by coimmunoprecipitation.     

Identification of two major virion protein genes of white spot syndrome virus of shrimp

White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 x 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26 and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26 (612) and vp28 (612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.     

Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.     

Identification of white spot syndrome virus (WSSV) envelope proteins involved in shrimp infection

White spot syndrome virus (WSSV) is a major shrimp pathogen causing large economic losses. In an attempt to identify the envelope proteins involved in virus infection, antisera against six WSSV envelope proteins were used in neutralization assays conducted in vivo. The results showed that the virus infection could be significantly delayed or neutralized by antibodies against three WSSV envelope proteins (VP68, VP281 and VP466). This neutralization was further confirmed by quantitative PCR. It could be concluded that the viral envelope proteins VP68, VP281 and VP466 played roles in WSSV infection to shrimp.     

White spot syndrome virus envelope protein VP28 is involved in the systemic infection of shrimp.

White spot syndrome virus (WSSV) is a large DNA virus infecting shrimp and other crustaceans. The virus particles contain at least five major virion proteins, of which three (VP26, VP24, and VP15) are present in the rod-shaped nucleocapsid and two (VP28 and VP19) reside in the envelope. The mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon, and the role of these proteins in these processes are not known. A specific polyclonal antibody was generated against the major envelope protein VP28 using a baculovirus expression vector system. The VP28 antiserum was able to neutralize WSSV infection of P. monodon in a concentration-dependent manner upon intramuscular injection. This result suggests that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic WSSV infection in shrimp.     

Multiple envelope proteins are involved in white spot syndrome virus (WSSV) infection in crayfish

Summary. White spot syndrome virus (WSSV) is a devastating viral pathogen of cultured shrimp worldwide. Previous studies have shown that the intact virion consists of at least 39 structural proteins and, among them, six were identified as envelope proteins involved in the virus infection. In this paper, the structural proteins VP36A, VP36B and VP31 (J Virol 2004; 78: 11360–11370), containing the RGD motif, were expressed in Escherichia coli and used to produce specific antibodies. Western blot confirmed that VP36A is a newly reported envelope protein. A neutralization assay with these three antibodies demonstrated that VP36A, VP36B and VP31 could significantly delay the initial infection of crayfish, but mortality still reached 100% at day 11 post-injection. However, a neutralization assay with the combination of antibodies against different envelope proteins showed that a combination of VP36B and VP31 antibodies could strongly inhibit WSSV infection in crayfish. These results revealed that multiple envelope proteins are involved in WSSV infection in crayfish and that VP36B and VP31 play a key role during this process.     

Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31

Based on a combination of SDS-PAGE and mass spectrometry, a protein with an apparent molecular mass of 31 kDa (termed as VP31) was identified from purified shrimp white spot syndrome virus (WSSV) envelope fraction. The resulting amino acid (aa) sequence matched an open reading frame (WSV340) of the WSSV genome. This ORF contained 783 nucleotides (nt), encoding 261 aa. A fragment of WSV340 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with a 6His-tag, and then specific antibody was raised. Western blot analysis and the immunoelectron microscope method (IEM) confirmed that VP31 was present exclusively in the viral envelope fraction. The neutralization experiment suggested that VP31 might play an important role in WSSV infectivity.     

Development of a polyclonal antibody specific to VP19 envelope protein of white spot syndrome virus (WSSV) using a recombinant protein preparation

The VP19 gene encoding a structural envelope protein of white spot syndrome virus was cloned into an expression vector and introduced into E. coli. The objective was to produce a recombinant VP19 structural protein. After induction, the recombinant VP19 protein (rVP19) was produced, purified by SDS-PAGE and used for immunization of Swiss mice for polyclonal antibody production. The mouse anti rVP19 antiserum had specific immunoreactivity to the viral antigen in WSSV infected Penaeus monodon as verified by immunohistochemistry and Western blot. The production of monoclonal antibodies against this rVP19 may be useful in order to combine with anti-VP28 monoclonal antibodies for enhancing the sensitivity of various WSSV serological assays.     

DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus

The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300 kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.     

Beta-integrin mediates WSSV infection

White Spot Syndrome Virus (WSSV) is a virulent and widespread dsDNA virus with a wide range of hosts. Although remarkable progress has been made on virus characterization, however, its mechanism of infection is poorly understood. In this study, by analyzing the phage display library of the WSSV genome, a WSSV envelope protein VP187 (wsv209) was found to interact with shrimp integrin. VP187 possesses the RGD motif. The interaction between integrin and VP187 was confirmed with coimmunoprecipitation. These results demonstrate for the first time an interaction between the WSSV envelope protein and a cell surface molecule. Soluble integrin, integrin-specific antibody and an RGD-containing peptide were found to block the WSSV infection in vivo and in vitro. Gene silencing using a sequence-specific dsRNA targeting beta-integrin effectively inhibited the virus infection. These findings suggest that beta-integrin may function as a cellular receptor for WSSV infection.     

A vector that expresses VP28 of WSSV can protect red swamp crayfish from white spot disease

White spot disease caused by white spot syndrome virus (WSSV) leads to devastating losses in shrimp farming. The WSSV envelope protein VP28, can be used as subunit vaccines that can efficiently protect shrimp against WSSV disease. However, the function of the envelope protein VP19 was not confirmed, some researches found that VP19 could protect shrimp against WSSV, and other reports found it no any protection. To detect the functions of VP28 and VP19 and find a method to prevent this disease in red swamp crayfish Procambarus clarkii, we constructed the plasmid vectors pIevp28 and pIevp19, which contains the ie1 promoter and coding region of vp28 or vp19 of WSSV, respectively. The results of quantitative real-time PCR and western blot showed that the injected vectors could transcribe corresponding mRNAs and translate to the protein VP28 or VP19 in the crayfish. The vp28 or vp19 signal was detected on the third day post injection, and maintained its expression for 30 days. The mortality of the crayfish with pIevp28 showed obvious decline compared with the controls (pIe and PBS injection). However, pIevp19 seems did not affect the mortality of the crayfish compared with the controls. Furthermore, only VP28 was found tightly bound to the host haemocytes under immunocytochemistry. The results suggest that the VP28 protein might protect shrimp from the virus through competitive inhibition. We also found that oral administration of Escherichia coli with pIevp28 could protect crayfish from white spot disease, but the E. coli with pIevp19 was not. Therefore, we think that oral administration of bacteria with pIevp28 is a potentially easy therapeutic way against white spot disease in aquaculture.