Preliminary Study On The Mechanism Of Cofilin1 And Cofilin2 Inhibit The Infection Of White Spot Syndrome Virus In Cherax Quadricarinatus

White spot syndrome virus(WSSV)is a rod-shaped double-stranded DNA with a wide range of hosts,strong infectious ability and high lethality,which is the most harmful virus in shrimp farming industry over the world.Studies showed that WSSV can infect hosts via the actin-mediated endocytic pathway.Cofillin as an important factor in the regulation of cytoskeleton regulate the assembly of cytoskeleton by promoting actin depolymerization,and then affect the invasion of various pathogens.We have found that the expression of actin depolymerization factor(CqCofilin)was influenced by WSSV infection in Cherax quadricarinatus.However,it is unknow whether CqCofilin participates in the WSSV infection by regulating the depolymerization of actin.In this study,the mechanism of CqCofilin in WSSV infection was initially explored by the in vitro culture of CqCofilin in WSSV infection,determined by real-time quantitative PCR,recombinant protein expression,and pulldown assays,results as following:(1)Gene cloning of CqCofilin 1(GenBank No:MT552632)and CqCofilin2(GenBank No:MT552633).The full-length cDNA of CqCofilin1 was 1762 bp,including an open reading frame(ORF)of 444 bp that encoded 147 amino acids,a 5’-untranslated region(UTR)of 292 bp and a 3’-UTR of 1026 bp.The calculated protein molecular weight was 17 kDa with a theoretical isoelectric point(pI)of 4.96.The full-length cDNA of CqCofilin2 consisted of 1315 bp with a 444 bp ORF encoding a polypeptide of 147 amino acids,a 5’UTR of 72 bp,a 3’UTR of 799 bp.The calculated protein molecular weight was 16.9 kDa with a theoretical pI of 5.95.Both of them contain only one actin-depolymerizing factor homology domain(ADF).The CqCofilin1 and CqCofilin2 were respectively clustered closing to PtCofilin1 and PtCofilin2 from Portunus trituberculatus in the phylogenetic tree.(2)Tissue distribution showed that CqCofilin1 was highly expressed in nerve,and CqCofilin2 was highly expressed in muscle.The tissue distribution of CqCofilin1 and CqCofilin1 were analysed.It suggested that CqCofilinl and CqCofilin2 were found in all tissues tested.CqCofilin1 was highly expressed in nerve,hemocyte,Hpt,hepatopancreas and gill,while CqCofilin2 was highly expressed in hemocyte,muscle,nerve,and Hpt.(3)The entry and replication of WSSV were increased by gene silencing of CqCofilin1 or CqCofilin2.RNA interference(RNAi)assay showed that the entry of WSSV was significantly increased in Hpt cells after gene silencing of CqCofilin1 or CqCofilin2.Meanwhile,the transcript of IE1 was significantly up-regulated at 3,6 and 12 h,VP28 was up-regulated at 6 and 12 h.(4)The recombinant protein of CqCofilin1 and CqCofilin2 overloading decreased WSSV replication.The recombinant protein of CqCofilins(rCqCofilin1 and rCqCofilin2)with His-tag were expressed in E.coli(BL21:DE3),and purified using Ni Resin 6F beads.The transcription level of IE1 was significantly reduced at 3,6 and 12 h,and that of VP28 was significantly decreased at 6 and 12 h after transfection of rCqCofilin1 or rCqCofilin2 followed by WSSV infection in Hpt cells.(5)Recombinant CqCofilinl and CqCofilin2 proteins interact with Cqβ-actin.His pulldown assay demonstrated that rCqCofilin1 and rCqCofilin2 clearly interact with Cqβ-actin from hemocyte.This study confirmed that CqCofilin1 and CqCofilin2 inhibit the entry and transcription of WSSV by serving actin cytoskeleton,which provide a new evidence for clarifying the mechanism of WSSV infection.