Mechanism Of Resistance To WSSV Infection By Regulation Of PcSnail Expression In Procambarus Clarkii

Procambarus clarkii,commonly known as crayfish,is an important aquaculture species in China.With the yearly increase of the crayfish farming area,disease outbreaks have made farmers suffer serious economic losses.White spot syndrome virus（WSSV）infection has caused massive mortality in crayfish.The zinc finger protein Snail is highly expressed in many malignant tissues and promotes the spread and metastasis of cancer cells through epithelial-mesenchymal transition（EMT）.However,the role of Snail in animal resistance to viral infection remains to be further revealed.In this study,a Snail gene,named PcSnail,was identified from the crayfish,and the role of PcSnail in resistance to WSSV infection in crayfish was investigated.The main results are as follows.The full-length c DNA of PcSnail is 1484 bp,with an open reading frame of 1197 bp,encoding 398 amino acids.The result of phylogenetic tree analysis showed that PcSnail is closely related to the Snails of Penaeus monodon and Litopenaeus vannamei.The C-terminus of PcSnail protein contains five classical Cys2-His2（C₂H₂）-type zinc finger domains.And the multiple amino acid sequence comparison revealed that the zinc finger domain is highly conserved.Tissue distribution analysis showed that PcSnail was widely distributed in several tissues,with the higher expression in the gills and intestine.The transcriptional level of PcSnail showed a decreasing trend in both gills and intestine in 12 h of WSSV infection in crayfish.In order to investigate the function of PcSnail protein in the crayfish antiviral response,RNAi technology was used to interfere with PcSnail in crayfish,and then infected with WSSV.The results showed that the copies of WSSV in the gills and intestine were both decreased.Injected with the recombinant protein of PcSnail resulted in a significant increase in the copies of WSSV in the gills,indicating that PcSnail was involved in the infection or replication of WSSV in crayfish.To explore the role of PcSnail in the process of WSSV infection or replication,after injecting ds PcSnail,WSSV was used to stimulate crayfish to detect the nuclear translocation of Relish.The results showed that the level of Relish entering the nucleus was decreased.And the results were verified by immunocytochemistry experiments.After interfering with Relish,the expression level of the immediate-early gene ie1 of WSSV was decreased in crayfish.The transcription level of ie1 was significantly decreased after interfering with PcSnail.In summary,WSSV kidnapped the host PcSnail in an unknown way and induced the transcription of its immediate-early gene ie1 in the host by promoting the transcription factor Relish into the nucleus.The host responds to this escape mechanism of WSSV by reducing the expression of PcSnail in the early stage of WSSV infection.This study provides a new idea for the crayfish response to the immune escape of WSSV.