CqFlightless-Ⅰ Promotes The Depolymerization Of Cytoskeleton To Inhibit White Spot Syndrome Virus Infection In Cherax Quadricarinatus

White spot syndrome virus(WSSV)belongs to the Nimaviridae(genus Whispovirus)with a genome of about 300 kbp and is a rod-shaped or circular doublestranded DNA virus.WSSV has a wide range of hosts and is highly infectious and lethal to crustaceans,especially prawns.The rapid spread and high mortality of WSSV have long plagued the world shrimp aquaculture industry and caused huge economic losses.However,no effective ways and methods have been found to prevent and treat the infection of WSSV.Therefore,further research on the mechanism of WSSV infection will help to find effective ways to prevent and treat WSSV infection.In viral infection,the cytoskeleton plays an important role in the transcriptional assembly and release of progeny viruses and participates in viral infection by responding to different physiological states or by dynamic continuous polymerization,dissociation,and rearrangement processes.However,the specific role of cytoskeleton and its related factors in the process of WSSV infection is not fully understood.Previously,we found that the transcript of an actin-associated protein CqFlightless-Ⅰ was up-regulated in a differentially expressed transcriptome library of the hematopietic tissue(Hpt)cells from Cherax quadricarinatus post white WSSV infection.FlightlessI is a member of the actin remodeling protein gelsolin family,involved in the depolymerization and rearrangement of the cytoskeleton.However,the role of Flightless-Ⅰ in the infection process of WSSV has not been studied.In this study,DNA sequence of CqFlightless-1 open reading frame(ORF)was cloned from the genome of Cherax quadricarinatus,and achieved the following results:1.The encoding region of this gene CqFlightless-Ⅰ was 3870 bp,encoding 1290 amino acids,including 11 leucine-rich repeat(LRR)domains and 6 gelsolin-like(GEL)domains.The result of multi-sequence alignment showed that the amino acid sequence of CqFlightless-Ⅰ had high homology with animals from human to arthropod,and the homology with shrimp and crab was the highest.Phylogenetic tree construction indicated that CqFlightless-Ⅰ was more closely related to Flightless-Ⅰ of crustacean.Tissue distribution analysis showed that the mRNA transcript of CqFlightless-Ⅰ expressed in all tissues in Cherax quadricarinatus,with the highest expression in muscle,followed by hematopoietic tissue(Hpt)cells.2.CqFlightless-Ⅰ expression was obviously enhanced after WSSV infection of Hpt cells.In addition,the immediate early gene IE1 and late viral gene envelope protein VP28 of WSSV were significantly enhanced post WSSV infection after gene silencing of CqFlightless-1 in Hpt cells,indicating that CqFlightless-Ⅰ could weaken WSSV infection.3.Interestingly,pull down and co-immunoprecipitation experiments have shown that CqFlightless-Ⅰ protein interacted with cytoskeletal Cqβ-actin in Hpt cells.After CqFlightless-Ⅰ was overexpressed in HeLa cells,the cytoskeleton was observed by confocal microscopy and the cytoskeleton aggregation was significantly reduced,which means that the CqFlightless-Ⅰ may through the cytoskeleton depolymerized blocking WSSV transport in cells.4.Futhermore,the level of he endosomal associated protein CqVCP and CqRABGEF1 in Hpt cells increased significantly by gene sicliencing of CqFlightlessI,while the expression level of autophagy-related protein GABARAP did not significantly change,suggesting that the function loss of CqFlightless-Ⅰ may affect the endosomal transport pathway of WSSV,but not the autophagy degradation process.In conclusion,this study illustrates that CQFlightless I reduces the infection efficiency of WSSV through depolymerizing cytoskeleton,and at the same time reduces the endosomal transport of WSSV,which provides a new theoretical reference for further searching for prevention and control of WSSV infection.