Mechanisms Of DNAJA3 And RNF5 Inhibiting Foot-and-mouth Disease Virus Replication

Foot-and-mouth disease(FMD)is an acute and highly contagious disease caused by foot-and-mouth disease virus(FMDV)in cloven-hoofed animals such as cattle,sheep and pigs etc,which has seriously jeopardized the world's livestock industry,social politics and economy for more than 100 years.Therefore,it has been listed as a statutory disease by the World Organisation for Animal Health(OIE)and classified as a first-class animal disease in China.The FMDV structural protein VP1 is exposed on the surface of the viral capsid and contains the major antigenic determinant and receptor binding site RGD,which plays an important role in virus-induced antibody response,adsorption and intervention.However,the interaction between VP1 and host protein and their mechanisms for regulating FMDV replication remains unclear.Therefore,this study screened the host protein DNAJA3 and E3 ubiquitin ligase RNF5 interacting with FMDV VP1 and elucidated the mechanism behind inhibition of FMDV replication,and the details are as follows:(1)Mechanism of host protein DNAJA3 inhibiting FMDV replicationIn this study,the host protein DNAJA3 binding to VP1 was identified by yeast two-hybrid technique.Co-immunoprecipitation(CO-IP)and confocal experiments confirmed the endogenous interaction between DNAJA3 and VP1.The results of the plasmid truncation assay and the alanine scanning assay indicated that the J domain of DNAJA3 [amino acid(aa)1–168] and the lysine 208(K208)site of VP1 were critical for the DNAJA3-VP1 interaction.Overexpression of DNAJA3 dramatically dampened FMDV replication,whereas silencing of DNAJA3 elicited an opposite effect against FMDV replication.Construction of the K208 A mutant recombinant virus was used to further confirm that the K208 site of VP1 was critical for DNAJA3-induced viral titer reduction.Further mechanistic studies revealed that DNAJA3 induced lysosomal degradation of VP1 by interacting with LC3 to enhance the activation of lysosomal pathway.Meanwhile,we discovered that VP1 suppressed the IFN-? signaling pathway by inhibiting the phosphorylation,dimerization and nuclear translocation of IRF3.Overexpression of DNAJA3 significantly attenuated VP1-mediated inhibition of the IFN-? signaling pathway,and this inhibition was significantly enhanced in DNAJA3 knockout cells.Poly(I:C)-induced IRF3 phosphorylation was also decreased in DNAJA3 knockout cells compared with DNAJA3 normal cells.(2)Mechanism of E3 ubiquitin ligase RNF5 inhibiting FMDV replicationThis study found that FMDV infection could up-regulate E3 ubiquitin ligase RNF5 both in vitro and in vivo,and the results of overexpression assay and siRNA interference assay indicated that RNF5 inhibited FMDV replication depending on its E3 ubiquitin ligase activity of C-cysteine 42.Further studies showed that RNF5 specifically degraded VP1 via the proteasome pathway.Co-IP assays showed that RNF5 mediated its polyubiquitination degradation by directly interacting with FMDV VP1.In addition,RNF5 not only degraded FMDV VP1,but also degraded VP1 of PV,SVA and EV71,and the degradation process depended on the E3 ligase activity of RNF5.In conclusion,this study revealed a novel function of the host protein DNAJA3 in antiviral responses,which degraded VP1 by inducing the lysosomal pathway and impaired the inhibitory effect of VP1 on the IFN-? signaling pathway;and clarified the mechanism of E3 ubiquitin ligase RNF5 inhibiting FMDV replication by ubiquitination degradation of VP1.Meanwhile,RNF5 had a broad-spectrum antiviral effect on Picornaviridae.Our research provided a theoretical basis for designing efficient vaccine candidate strains(or antigens).