WSSV Escapes From Autophagic Elimination Via Endosomal Trafficking Facilitated By CqVCP

As the most severely lethal viral pathogen for crustaceans both in brackish water and freshwater,white spot syndrome virus(WSSV)has caused a severe ecological problem for aquaculture areas around the world.WSSV is the sole member of the Whispovirus genus in the Nimaviridae family,which is composed of envelope,nucleocapsid and double-stranded DNA.Heretofore,studies on the endocytosis process of host cells infected with WSSV have been revealed,but the subsequent intracellular transport process and penetration of WSSV are unclear.Here,hematopoietic tissue(Hpt)stem cells from red claw crayfish C.quadricarinatus were used as the research model to observe the stage of vacuolar trafficking of internalized WSSV and analyze the function and key regulatory mechanism of this process,in order to provide an ideal target for anti-WSSV infection.Firstly,it was found that the internalized WSSV were delivered into the endosomal system through endocytic vesicles,and then accompanied with the dynamic transport of the endosomes,the envelope of WSSV fused with endosome membrane and its nucleocapsid release into the cytoplasm.The dynamic transport of endosomecontaining WSSV in vitro for the first time was monitored by using real-time confocal technique.The ultrastructural analysis demonstrated that envelope fusion of endocytosed WSSV drove nucleocapsid release.The inhibition on endosomal acidification significantly restricted the endosomal escape of WSSV genome,resulting in retention of WSSV in alkalized endosomal compartments.Meanwhile,inhibition on envelope dissociation of endocytic WSSV obviously decreased the expression of viral late gene.These results suggested that endosomal alkalinization restricted the intracellular transport of internalized WSSV and subsequently prevented the transmembrane transmission of genomes involved in the nucleocapsid.According to this mechanism,ethier alkalizers Chloroquine and NH4Cl or vacuolar V-ATPase inhibitor Bafilomycin A1 could notably delayed endosomal transport of WSSV.However,inhibiting PIKfyve activity,participated in formation of muti-vesicular bodies(MVBs),could not effectively block disassembly and subsequent replication of WSSV.Hence,the acidic environment within early endosome was a prerequisite for the release of viral nucleocapsid into cytoplasm driven by envelope fusion,which facilitated successful transmembrane delivery of viral genome into cell nucleus.Secondly,it was found that a valosin-containing protein in C.quadricarinatus(CqVCP)promoted the penetration of WSSV from limiting endomembrane into cytoplasm.Interestingly,WSSV infection caused higher expression of CqVCP in vivo.Meanwhile,gene interference or pharmacological inhibition of CqVCP apparently decreased viral replication by blocking intracellular transport in vitro.Remarkably,the binding of an ATP-competitive inhibitor DBeQ with CqVCP prevented nucleocapsid release from endosome into cytoplasm by disrupting endosomal acidification.Consisted with delivery of endocytosed WGA,lots of internalized WSSV virions were limited in alkalized endosomal compartment in DBeQ-treated Hpt cells.Meanwhile,DBeQ treatment caused co-localization of RabGEFl and CqVCP with WSSV detained into damaged endosome.TEM analysis showed that multiple intact WSSV virions were segregated together so as to generate viral aggregation by blocking the ATPase activity of CqVCP.Differently,another allosteric VCP inhibitor NMS873 blocked delivery of endocytic WSSV to endosome by hampering fusion of endocytic vesicle with endosome.Furthermore,both DBeQ and NMS873 obviously prohibited nuclear replication and assembly of progeny WSSV by pre-inhibiting intracellular trafficking of WSSV.Additionally,inhibition on CRLs-proteasome with inhibitor MLN4924 and MG 132 didn’t prevent endosomal escape and replication of WSSV effectively,which indicated that the role of CqVCP played in proteasome-mediated degradation couldn’t been involved in endosomal acidification.Therefore,the dysfunction of CqVCP not only inhibited delivery of endocytic vesicles to endosome,but also effectively detained invading WSSV into injured endosome via disrupting endosomal acidification in WSSV-infected Hpt cells.Thirdly,it was found that damaged endosome-mediated autophagy would be used as an effective strategy to segregate and eliminate WSSV detained into endosome.DBeQ promoted lipidation of autophagy marker CqGABARAP in a dose-dependent manner,which subsequently decorated WSSV/WGA-localized endosome in Hpt cells.However,the initiation of autophagy caused by DBeQ was significantly prohibited after pretreatment with NMS873 and Bafilomycin Al,indicating that endosome-mediated autophagy was regulated by CqVCP-V-ATPase pathway in Hpt cells.Moreover,the CqVCP-V-ATPase pathway may also contribute to endosomal acidification for promoting endosomal escape of WSSV.In addition,the lipidation of CqGABARAP was promoted by AKT inhibitor AKTi 1/2 in dose-dependent manner,which subsequently decorated WSSV/WGA-localized endosome in Hpt cells.Similar to DBeQ,AKTi 1/2 treatment restricted viral disassembly to form WSSV aggregates in Hpt cells.Unlike DBeQ,AKTi 1/2 treatment enhanced endosomal acidity and cathepsin activity to degrade segregated WSSV.By comparing the isolation effect of endocytic WSSV,AKTi 1/2 treatment was strikingly weaker DBeQ treatment and there was no additive effects when they were added up.Whereas NMS873 pretreatment prohibited endosomal accumulation and acidification in AKTi1/2-treated Hpt cells.These results suggested that AKT may be upstream of CqVCP in endosome-mediated autophagy,which benefited endosomal escape of endocytosed WSSV.In summary,endosome system promoted WSSV infection during intracellular trafficking,while autophagy acted as a host defensive mechanism against WSSV infection.Especially,the fusion of viral envelope with early endosomal membrane was prerequisite for transmembrane delivery of WSSV genome in WSSV-infected Hpt cells.Among this,CqVCP-V-ATPase signaling pathway made sure nucleocapsid release via maintaining endosomal acidification.Moreover,AKT and CqVCP promoted endosomal escape of WSSV genome to avoid host defensive autophagy.Consequently,activating endosome-mediated autophagy restricted endosomal escape of WSSV by targeting against AKT-CqVCP sianaling pathway,which would provid an important anti-WSSV strategy in early-phase infection of WSSV.