Identification And Characterization Of The Binding Proteins Of VP31and VP110in Chinese Shrimp, Fenneropenaeus Chinensis

White spot syndrome virus (WSSV) belongs to the genus Whispovirus of the family Nimavirridae, which is highly virulent to penaeid shrimp and has caused serious economic losses to shrimp farmers throughout the world. WSSV is an enveloped virus with a double-stranded circular DNA genome of about300kb. It is known that the viral envelope proteins play very important roles in WSSV infection in shrimp, such as recognition and attachment to receptors at the host cell surface. In recent years, several researches concerning interactions between the viral envelope proteins and host cells were performed and provided some valuable information about WSSV infection mechanism. Previous study showed that the polyclonal antibodies against VP31had a WSSV-neutralizing activity, VP110was capable of attaching to host cells and adhesion could be inhibited by synthetic RGDT peptides, which suggested that VP31and VP110play important role in WSSV infection. However, up to now, there is no report about the interaction between VP31and host cell, and the functions of VP31and VP110involved in WSSV infection is still unclear. In present work, the VP31and VP110was recombinantly expressed, and employed as a probe to identify the VP31-binding proteins in hemocytes of F. chinensis by virus overlay protein binding assay (VOPBA), and VP110-binding proteins in hemocyte and gill of F. chinensis by pull down and VOPBA, then the VP31-binding protein and VP110-binding protein were identified and recombinantly expressed for further investigation of its potential roles in WSSV infection by in vivo neutralization assay. The followings are the details:To investigate the interaction between WSSV envelope protein VP31and hemocyte of Chinese shrimp, F. chinensis, the VP31protein was recombinantly expressed in Escherichia coli as a fusion protein with GST tag. By virus overlay protein-binding assay (VOPBA), a prominent protein band of26kDa in hemocytes of F. chinensis was recognized by the recombinant VP31(rVP31), which was identified as triosephosphate isomerase (TPI) by mass spectrometric (MS) analysis. Then, the TPI gene of F. chinensis was cloned and expressed as a fusion protein with Trx/His/S-tag using pET-32(a)+vector, and the binding interaction between the recombinant TPI (rTPI) and rVP31was further confirmed by enzyme-linked immunosorbent assay (ELISA) and pull-down assay. TPI mRNA level was the highest in goand and gut was very low, and TPI mRNA levels in hemocytes was down-regulated to approximate1/2compared to the control group post WSSV infection by real-time RT-PCR. In vivo neutralization assay, the rTPI appeared to be able to partially block the WSSV infection and delay the death of infected shrimp. These results suggested that TPI in F chinensis might play a potential role in WSSV infection.A segment of VP110, which include an forkhead associate domain (FHA) peptides, was amplified and inserted into pET-32(a)+vector and expressed as a fusion protein with Trx/His/S-tag in E. coli. BL21(DE3). Recombinant VP110protein (rVP110) was used as a bait protein in pull-down assay, and two bands protein were preyed from gill protein of F. chinensis, meanwhile, two protein bands from gill protein of F. chinensis were detected that could react with rVP110by VOPBA. One band protein was preyed from hemocyte of F. chinensis by VOPBA and pull down assay. Three binding proteins were excised and analyzed by MS, which were ariginine kinese (AK) and β-actin respectively. Previous study showed that β-actin play an import role in WSSV infection, to clarify the interaction between VP110and AK, recombinant AK protein (rAK) was obtained by prokaryotic expression. Meanwhile, the interaction between rVP110and rAK was analyzed by using ELIS A and pull down assay, the result showed rVP110could bind to the shrimp gill proteins, hemocyte protein and rAK. AK mRNA was the highest level in muscle and very low in gut, and AK mRNA levels in gills was up-regulated compared to the control group post WSSV infection by real-time RT-PCR. Moreover, experiment in vivo neutralization experiment was carried out, which showed rAK could inhibit WSSV infecting shrimp. These results suggest that VP110is involved in WSSV infecting shrimp.