The Role And Molecular Mechanism Of Wnt5b And Tcf In Litopenaeus Vannamei During WSSV Infection

Litopenaeus vannamei culture industry has always been restricted by the diseases caused by viruses.Among these pathogens,white spot syndrome virus(WSSV)is the most fatal and virulent one,which has caused substantial economic losses.Up to date,there is no effective method to control the virus.Therefore,understanding the mechanisms of the host immunity and the host-virus interaction is of great importance to find new means for WSSV control.Wnt signaling pathway is highly conserved among different species,it also play an important role in immune regulation.In this work,we cloned and characterized a Wnt5b homolog form L.vannamei(designated as LvWnt5b).qRT-PCR showed that the transcriptional level of LvWnt5b was down-regulated after WSSV infection.Silent the expression of LvWnt5b by RNAi resulted in the lower expression of WSSV immediately-early gene iel.At the same time,caspase3/7 activity in shrimp hemocytes was increased,which indicate that LvWnt5b mediated Wnt signaling pathway contributed to inhibit the cell apoptosis.Moreover,overexpression of LvWnt5b in HEK293T cells led to inhibition of caspase3/7 activity,which further proved the role of LvWnt5b in restraining apoptosis.All these results showed that the shrimp decreased the expression of LvWnt5b initiatively to against WSSV infection via promoting apoptosis.Nuclear DNA-binding TCF proteins,acting as the main downstream effectors of Wnt signaling pathway,are vital to cell-fate decisions during embryonic development and in adult tissues.We obtained the full-length of LvTcf from L.vannamei transcriptome which was analyzed in our lab.Then its roles in WSSV infection were analyzed.qRT-PCR showed that the expression of LvTcf significantly increased during WSSV infection.Knock-down of LvTcf was beneficial to the expression of ie1,indicating that LvTcf played a positive role in WSSV infection.In cells,the expression of target genes would be induced after β-catenin combining with TCF.Co-immunoprecipitation experiments indicated that Lvβ-catenin interacted with LvTcf.Immunofluorescence assay further showed that Lvβ-catenin and LvTcf had a strong colocalization in Sf9 cells.Our further research performed by dual-luciferase reporter assay showed that LvTcf could increase the expression of interferon-like gene Vago1 in shrimp.As a pivotal nuclear co-activator of Wnt signaling,TCF is regulated by various host and microbe molecules.WSV083,a serine/threonine protein kinase of WSSV,was found to bind to and phosphorylate LvTcf Phosphorylated LvTcf was then degraded via ubiquitin-protosome pathway in cytoplasm.Further,mass spectrometric analysis was utilized to locate T39 and T104 as the extract amino acid residues of LvTcf which were phosphorylated by WSV083.However,point mutation analysis suggested that more sites except T39 and T104 of LvTcf could be phosphorylated by WSV083.In sum,our study elucidated the role of L.vannamei Wnt5b and Tcf during WSSV infection.At the same time,we also illustrated the role in immune regulation of LvTcf and the mechnasim that it was regulated by WSSV,which could provide new ideas to find the way for WSSV control with theoretical support.