Mechanism Of PcTrim And ATF4-β In Against White Spot Syndrome Virus Infection In Procambarus Clarkii

Viral diseases are a perennial topic.The virus not only causes huge economic losses to the farming industry,but also endangers human life and health.Due to the diversity of viral infections and its extreme variability,the prevention and treatment of the virus is extremely difficult at present,so the autoimmune system plays a key role in resisting virals infection.White spot syndrome virus(WSSV),a virulent pathogen,is a circular double-stranded DNA virus,has the largest genome(300 kb),casused serious economic losses.Crustaceans as important aquaculture species can create huge economic benefits for aquaculture industry every year.Invertebrates only resist infection by innate immunity,which is a good model to study innate immunity.WSSV was used as a model to study the mechanism by which the host inhibits the entry of the virus into cells at early stage and resists virus infection at late stage,providing an effective basis for the prevention and treatment of the virus.1.PcTrim prevents early infection of white spot syndrome virus by inhibiting AP1-induced endocytosisTripartite motif(TRIM)family proteins have been reported to be associated with phagocytosis and weres also believed to be involved in the activation of autophagy.WSSV can enter host cells through autophagy.The role of TRIM in the early stage of virus infection in host cells remains to be further interpreted.In the current study,a crayfish TRIM with a RING-type domain,designated as PcTrim,was significantly upregulated under white spot syndrome virus(WSSV)infection in the red swamp crayfish(Procambarus clarkii).Recombinant PcTrim(r PcTrim)significantly inhibited WSSV replication in crayfish.RNAi or blocking PcTrim with an antibody promoted WSSV replication in crayfish.Pulldown and co-IP assays showed that PcTrim can interact with the virus protein VP26.PcTrim restricts the expression level of dynamin,which is involved in the regulation of phagocytosis,by inhibiting Activating protein 1(AP1)entry into the nucleus.AP1-RNAi effectively reduced the expression levels of dynamin and inhibited host cell endocytosis of WSSV in vivo.Our study demonstrated that PcTrim might reduce early WSSV infection by binding to VP26 and then inhibiting AP1 activation,resulting in reduced endocytosis of WSSV in crayfish hemocytes.2.ATF4-β depends on HSP70 and HMGBa to enter the nucleus and regulate the expression of ALFs to participate in antiviral immunityActivating transcription factor 4(ATF4)is a member of the ATF/CREB family of transcription factors.While pivotal roles for ATF4 have been discussed in endoplasmic reticulum(ER)stress and metabolic regulation,there are few reports investigating its role in the antiviral immune response.The nuclear translocation mechanism of ATF4 remains incompletely understood.We identified two isoforms of ATF4 with a difference of only 7 amino acids,and they were designated ATF4-α and ATF4-β.Through the structural models of ATF4-α and ATF4-β,we found that ATF4-β has one less β-fold structure than ATF4-α.Further studies showed that only the nuclear translocation of ATF4-β increased significantly after virus infection.Through Co-IP assays,we demonstrated that Heat shock protein 70(HSP70)and High mobility group protein a(HMGBa)only bind to ATF4-β but not ATF4-α,further indicating that ATF4-β plays a more critical role in antiviral immunity than ATF4-α.When WSSV infects organisms,HMGBa located on the membrane recognizes the WSSV envelope protein VP28 and then recruits HSP70 and ATF4-β to form HMGBa-HSP70-ATF4-β trimers,which in turn mediate the nuclear translocation of ATF4-β.After entering the nucleus,ATF4-β initiates the transcription of ATF4 and the transcription of the antilipopolysaccharide factor(ALF): ALF1 and ALF2.Then,ALFs are released extracellularly,and ALF1 can specifically bind to VP28 to inhibit virus assemble and reinfection.In this study,we elucidated the nuclear translocation mechanism of ATF4-β and clarified its role in the antiviral immune response,which provides novel insights into invertebrate antiviral immunity.