The Molecular Mechanism Activating Selective Autophagic Degradation Of PRV VP16 By Inhibition Of USP14

Herpesvirus alpha(α)is an envelope double-stranded DNA virus with unique latent reactivation mechanism and wide host spectrum.HSV-1/2,Varicella Zoster Virus(VZV),Pseudorabies Virus(PRV),Duck Enteritis Virus(DEV),and so on,which may infect humans and animals.Posttranslational modification refers to the covalent modification of amino acids after protein synthesis,including ubiquitination,phosphorylation,glycosylation and methylation.Posttranslational modifications play important roles in protein structure,stability,activity,and cellular localization.The ubiquitination of proteins involves three processes: E1,E2 and E3 that activate,bind,and bind ubiquitin to substrate proteins.The reverse reaction is known as de ubiquitination,and it is catalyzed by ubiquitinases(DUBs),which play important roles in immunity and infection.In order to study the role of USPs in the replication of alpha herpesvirus,13 USPs inhibitors were used to investigate the PRV replication.It was found that 13 types of USPs showed inhibitory effects to PRV-GFP,and USP14 inhibitor b-AP15 was the most prominent.Furthermore,CRISPR-Cas9 inhibits the replication of PRV.The expression of wild type USP14 could restore PRV replication in USP14 knockout cells,however USP14 deubiquitinase activity could not restore the replication of PRV,suggesting that USP14 deubiquitinase activity plays an important role in the replication of PRV.To study the mechanism by which USP14 can promote PRV proliferation,we studied the effects of b-AP15 on specific life cycle of PRV.Results showed that b-AP15 had no effect on PRV adsorption or invasion,while PRV replication was inhibited obviously.Further research found that treatment with b-AP15 for 10 minutes significantly inhibited transcription of PRV IE180,EP0 and UL9(early gene)genes.As PRV VP16 envelope proteins are involved in immediate and early transcription of PRV genes,this suggests that b-AP15 may promote ubiquitination and degradation of VP16.These results show that inhibition of USP14 promotes ubiquitination in 168 lysine(K168)of VP16.During early stages of infection with PRV,USP14 binds directly to K63 junction at K168 position of VP16 via its UBL domain,and then deubiquitinates VP16,ensuring early gene transcription necessary for virus replication.To identify specific pathways through which inhibition of USP14 promotes VP16 degradation,B AP15 combined with proteasome inhibitor MG132 as well as autophagy inhibitor 3-MA were used in this study.The results showed that MG132 had no effect on the degradation of VP16 induced by b-AP15,but 3-MA inhibited the degradation of VP16 induced by b-AP15,suggesting that inhibition of USP14 promotes the degradation of ubiquitination VP16 mediated by autophagy.Further studies have found that SQSTM1/p62 selective autophagic receptor binds directly to the K63 ubiquitin chain in VP16 K168 via its UBA domain,and is degraded through autophagic lysosomal pathway.Subsequently,this study examined whether inhibition of USP14 could activate intracellular autophagy.The results of immunofluorescence and transmission electron microscope show that USP14 can activate autophagy.Inhibition of key autophagic genes ATG5,BECN1 inhibits b-AP15 induced VP16 degradation,further demonstrating that inhibition of USP14 promotes autophagic VP16 degradation.To reveal a molecular mechanism through which inhibition of USP14 promotes autophagy,HSPA5/BiP,ATF6 and EIF2AK3 were investigated in this study.It was shown that inhibition of USP14 activated EIF2AK3 endoplasmic reticulum stress pathway and did not affect HSPA5/Bi P,ATF6 pathway.The inhibition of EIF2AK3 endoplasmic reticulum significantly inhibits autophagy and VP16 degradation,suggesting that inhibition of USP14 activates autophagy and VP16 degradation via EIF2AK3 stress pathway.Finally,we evaluated the preventive and therapeutic effects of b-AP15 against PRV infection in mice.After injection of b-AP15 into mice after PRV infection,it was found that b-AP15 could not only inhibit PRV replication in mice,but also inhibit inflammatory infiltration caused by PRV,which significantly improved the survival rate of mice.All in all,the mechanism of inhibiting selective autophagic degradation of pseudorabies virus VP16 activated by USP14 in order to provide a new theory basis for PRV prevention.