The Molecular Mechanism For Antagonism Of Interferon Production By Rabies Virus P Protein

Rabies is a zoonotic disease resulting from rabies virus(RABV)infection,which kills more than 59,000 people worldwide each year.Rabies has an almost100% mortality rate once it develops,and there is still no effective treatment.The genome released by RABV is mainly recognized by the pattern recognition receptor RIG-I in cells,which recognizes viral RNA and activates downstream signaling pathways to stimulate host innate immune responses and induce adaptive immunity.The RABV genome encodes five structural proteins,which are the N,P,M,G and L proteins.Of these,the P protein is multifunctional and plays an essential part in viral replication and innate immune escape.Previous studies have revealed that the P protein the major type Ⅰ IFN antagonist in viruses by both antagonizing host interferon(IFN)production and inhibiting IFN-mediated signaling pathways.However,the exact molecular mechanism and precise site of P protein antagonism of type Ⅰ IFN production is still not clear.Previous studies in our laboratory have demonstrated that infection with fixed and wild strains of RABV differ in their ability to activate the IFN pathway.In this study,we found that the P proteins of CVS and DRV differed in their ability to inhibit IFN activation.Based on hydrophobic analysis and region swapping between CVS-P and DRV-P,we found that the 172-182 amino acid region on DRV-P was essential for its inhibition of IFN.We then performed sequence alignment and site mutation on the 172-182 amino acid region of the two P proteins.It was found that serine(Ser)at position 179 was responsible for the inhibition of IFN production.The ability of P protein to inhibit the activation of IFN regulatory factor 3(IRF3)was significantly reduced when site 179 was mutated from Ser to proline(Pro).A protein interaction screen of key molecules in the RIG-I pathway revealed that RABV-P(hereafter named S179-RABV-P)with Ser at site 179 could bind to IKKε(I-kappa B kinase ε),a key kinase in the RIG-I pathway.Regional interaction experiments have shown that S179-RABV-P interacts with both the kinase domain(KD)and the scaffold dimerization domain(SDD)of IKKε.Subsequent studies have revealed that S179-RABV-P disrupts the binding of IKKε/IKKε-KD to the upstream molecule TRAF3 and IKKε/IKKε-KD to the downstream IRF3 by interacting with IKKε,thereby inhibiting IRF3 activation and interferon production.The recombinant mutant viruses r CVS-P179 S and r SAD-S179 P were obtained by reverse genetic modification of the P protein 179 site in CVS and SAD strains to Ser and Pro,respectively.Cellular assays showed that the 179 site Ser determined the interaction of the P protein with IKKε in infected cells and the ability of the virus to inhibit IFN in infected cells.Pathogenicity tests suggested that the alteration of the 179 site of P protein significantly affected the ability of RABV to inhibit host IFN activation.Compared to the parental strain,r CVS-P179 S challenged mice exhibited earlier onset of disease and increased symptoms;r SAD-S179 P was dramatically weakened in mice and the immunogenicity was not compromised.We also found that S179-P in other Lyssaviruses could also inhibit IRF3 activation by binding IKKε.In summary,this study identified Ser at 179 site of RABV-P as a key site for antagonizing IFN production.S179-RABV-P inhibits the IFN response by binding to the IKKε kinase molecule and disrupting the interaction of IKKε with TRAF3 and IRF3,and 179 site mutation of P protein can significantly alter the pathogenicity of the RABV.More importantly,the function of S179-P in antagonizing IFN production by targeting IKKε is conserved in Lyssavirus.