Molecular Mechanism Of Rabies Virus Replication Regulated By Viral Phosphoprotein

Rabies virus (RABV), belonging to the genotype 1 lyssavirus in the Rhabdovidae family, is the causative agent of human and animal rabies in the world. Currently there are seven major genotypes of Lyssavirus genus:genotype 1 (rabies virus, RABV), genotype 2 (Lagos bat virus, LBV), genotype 3 (Mokola virus, MOKV), genotype 4 (Duvenhage virus, DUW), genotype 5 (European bat Lyssavirus 1, EBLV-1), genotype 6 (European bat Lyssavirus, EBLV-2), and genotype 7 (Australian bat Lyssavirus, ABLV), among them RABV exhibits the greatest risk to humans and animials. The virus phosphoprotein (P) is a multifunctional nonkinase protein that is involved in viral transcription and replication, and in addition, RABV P is an interferon antagonist. Until now, while many studies have verified the interactions of P and other RABV virus structural proteins as well as its interactions with host cell proteins, detailed data on the interactions of P and host cell proteins is still limited, and the pathogenic mechanism of RABV is still far from clear. In this study, we illuminate the molecular mechanism of Cdc37 stabilizing P protein through Hsp90 binding-dependent and independent pathway to facilitate RABV replication, the purpose of which is to provide the basic reference data for developing novel drug targets for treatment and prevention of RABV infection.1. Analysis of RABV P as a client protein of Hsp90Our laboratory has previously discovered that the cytoplasmic Hsp90 is recruited into the site of viral transcription and replication called Negri Bodies (NBs) during RABV infection, suggesting that Hsp90 may be involved in RABV infection. We in this study choosed the mouse neuroblastoma N2a as a cell model of RABV infection and choosed the RABV vaccine strain HEP-Flury as a major virus strain used in the following experiments, then firstly analyzed the effect of Hsp90 inhibitors on RABV infection. The results of western blotting analysis showed that the expression level of virus proteins such as N and P in GA or 17-AAG-treated cells was significantly reduced compared to vehicle control; The results of RT-qPCR analysis found that viral RNA synthesis, including the level of viral mRNA, viral genome RNA and viral anti-genome RNA, was also severely decreased after Hsp90 inhibitors treatment; Meanwhile, the results of TCID50 assay showed that treatment with GA or 17-AAG also significantly suppressed virus production in virus infected N2a cells. We analyze the accumulation of RABV N mRNA by comparative quantitative RT-qPCR and the results demonstrated that treatment with 17-AAG do not markedly affect the N mRNA level in the presence of the de novo protein synthesis inhibitor cycloheximide (CHX) in RABV infected N2a cells. These results suggested that the RABV infection affected by Hsp90 was due to the stability of RABV virus proteins rather than virus transcription or the stability of viral mRNA directly regulated by Hsp90.However, how was the stability of RABV virus proteins affected by Hsp90? Whether Hsp90 affected the stability of RABV virus proteins through the proteasome and autophagy pathway? To examine this, the results of western blotting analysis in this study showed that the expression level of N or P was not increased but reduced in the presence of 17-AAG after treatment with the preteasomal inhibitor MG-132; However, RABV virus proteins including N and P substantially accumulated after treatment with the autophagy inhibitor wortmannin in the presence of 17-AAG. These results suggested that RABV virus proteins degradation induced by 17-AAG might occur through autophagy. To confirm the speculation of autophagic degradation of RABV virus proteins elicited by Hsp90 inhibiton and rule out the possibility of spurious off-target effects of wortmannin, we found that the level of N and P was significantly increased in the presence of 17-AAG after down-regulation of the key autophagy pathway component LC3B by shRNA, suggesting that Hsp90 inhibitors triggered autophagic degradtion of N and/or P protein. Meanwhile, we constructed the recombinant vectors pSG5 containing gene P or N and transfected these constructs into N2a cells. When these transfected cells were treated with 17-AAG, viral protein P but not N was markedly degraded. Furthermore, the results of Co-IP assay showed that the native or exogenously expressed RABV P protein interacted with endogenous or exogenously expressed Hsp90. The results of Co-IP assay also showed that Hsp90 strongly associated with all the three selected lyssavirues (CVS-11, ABLV and MOKV) P protein. The Hsp90-P interaction is a common property shared by all lyssaviruses, suggesting that all the lyssaviruses P protein is a client protein of Hsp90.2. Analysis of the formation of ternary Hsp90-Cdc37-P complexesTo accomplish the chaperone activity of Hsp90, Hsp90 molecular chaperone machinery requires a lot of cochaperone. To find whether there were some crucial Hsp90 cochaperones required for Hsp90-P interaction, we first used the known Hsp90 allosteric inhibitor Celastrol to investigate its effect on RABV virus proteins accumulation. The results of western blotting analysis showed that celastol reduced P protein accumulation as well as 17-AAG, suggesting that the cochaperone Cdc37 might be involved in the formation of Hsp90-P complex. Then we performed confocal microscopy with RABV infected N2a cells and the results showed that Cdc37 was co-localized with P protein in NBs. Furthermore, the results of Co-IP assay verified the formation of ternary Hsp90-Cdc37-P complexes, indicating that the P protein was a novel client protein of Hsp90/Cdc37 chaperone machinery.3. Analysis of Hsp90 binding-dependent and independent pathway of Cdc37 recruiting the P protein to Hsp90 machineryStudies have reported that Cdc37 have Hsp90 binding-dependent and independent pathway in the recruitment of kinases to Hsp90 machinery. However, little is known about the pathway of Cdc37 recruiting the nonkianse proteins to Hsp90 machinery. Whether there is the two pathway above or other pathway of Cdc37 in the recruitment of the nonkinase RABV P protein to Hsp90 machinery? In this study, we discovered four ways of Cdc37 involved the formation of Hsp90-P complex:The Cdc37 truncated mutants which lacks Hsp90 binding domain did not increased the level of P protein, while the wild type Cdc37 and Cdc37 truncated mutant(aa1-323) with Hsp90 binding-dependent activity in recruiting P to Hsp90 machinery promoted the accumulation of P protein; The Cdc37 mutants with single and double point mutations at residues M165 and L206 that showed greatly reduced binding to Hsp90 still associated with P and increased the level of P protein, and phenocopied wild type Cdc37 by increasing Hsp90-P association, indicating that Cdc37 recruits P to Hsp90 machinery in Hsp90 binding-independent manner; The activation of Cdc37 requires phosphorylation at Ser13. The inactive Cdc37 mutant(mutation of Serl3 to Ala), which did not compromise the recruitment of Cdc37 to Hsp90-P complex, also promoted the accumulation of P protein, and recruited P to Hsp90 as well as the wild type Cdc37, indicating that inactive Cdc37 also can recruit P to Hsp90 machinery in Hsp90 binding-dependent manner similarly to wild type Cdc37; The inactive Cdc37 mutants with single and double point mutations at residues M165 and L206 that showed greatly reduced binding to Hsp90 still associated with P and increased the level of P protein, and also phenocopied wild type Cdc37 by increasing Hsp90-P association, indicating that inactive Cdc37 recruits P to Hsp90 machinery in Hsp90 binding-independent manner similarly to wild type Cdc37.4. Analysis of positive regulation of RABV infection by Cdc37 and Hsp90 through stabilizing the P proteinFirstly, the results of western blotting analysis showed that RABV infection induced the cellular expression of Cdc37 and Hsp90, suggesting that Cdc37 and Hsp90 might play a positive role on the RABV infection. Secondly, the effect of exogenous overexpression of Hsp90 or Cdc37 on RABV infection was analyzed, and the results showed that Hsp90 and Cdc37 promote the accumulation of RABV P protein at protein level. Then the results of western blotting analysis showed that overexpression of Cdc37 or Hsp90 slowed the rate of P protein degradation but not N protein degradation following treatment with CHX, indicating that the stability or half-life of P protein is depended on Hsp90 and Cdc37.Additionally, down-regulation of Cdc37 or Hsp90 using RNA interference (RNAi) significantly decreased the level of viral proteins accumulation, viral RNA synthesis, and viral production, confirming the positive regulation of RABV infection by Cdc37 and Hsp90. Combined with the previous data related to Hsp90 and Cdc37 overexpression, Hsp90 and Cdc37 contribute to RABV infection by promoting P stability and increasing the accumulation of P.