The Effect Of Rabies Virus Infection In Vivo And In Vitro On The Differential Expression Of ApoD Gene And Its Interaction

Rabies is a zoonotic disease caused by the highly neurotropic rabies virus(RABV),which infect the central nervous system(CNS)and has 100% lethality.Although researchers have made fruitful achievements in long-term studies about RABV,but the pathogenic mechanism of RABV is still not fully elucidated.Apolipoprotein D(Apo D)is widely distributed in the heart,liver,spleen,kidney,skeletal muscle and brain.According to the brain proteomics study of rabies virus infected mouse,Apo D was up-regulated.ATP Synthase subunit-β(ATP5B)is located in the inner membrane of mitochondria and cell membrane,the Apo D protein interacting with ATP5 B was preliminarily screened by yeast two-hybrid system.In this study,we attempted to understand the interaction between RABV and Apo D and the correlation between Apo D and ATP5 B by exploring the expression patterns of Apo D in vitro and in vivo after RABV infection,which is helpful to understand and explore the pathogenic mechanism of RABV.Firstly,for the in vivo experiment,the mice were intracranially infected with attenuated r RC-HL,virulent GX074 and recombinant RABV mutants r RC-HL △ G,r RC-HL △ G242-288,and Mu11.Then the q RCR and Western Blot(WB)methods were used to detect the expression changes of viral proteins,host proteins Apo D and ATP5 B.The results showed that the m RNA and protein levels of Apo D were significantly up-regulated in the brains of all the virus-infected mice.At the 4 and 7 day post infection(dpi),the transcription level m RNA of Apo D gene was increased by 1.38-5.05 times and 3.48-8.25 times respectively,and the protein level of Apo D increased by 5.21-13.13 times and 7.01-15.35 times respectively.The change of ATP5 B was not significant in the infected mouse brain.Secondly,the effect of RABV infection on Apo D expression was detected in multiple cell line in vitro.BV2 cells were infected with 1 MOI virus,and the expression changes of Apo D and cytokines were studied by q PCR.The results showed that the expression of Apo D m RNA in the r RC-HL,Mu11 and CVS-11 infected BV2 cells was up-regulated by 2 times at 24 hpi,while the expression of Apo D protein was not significantly changed.At the early stage of r RC-HL and recombinant virus infection,high level of IFN-β expression was produced at 12 hpi.The expression of IFN-β m RNA was 27.43-53.29 times,and decreased to 9.02-29.43 times at 48 hpi.r RC-HL and CVS-11 can induce the expression of TNF-α m RNA up to 2 times at 24 hpi,which is consistent with the expression trend of Apo D m RNA.The standard virulent CVS-11 induced higher expression of IL-1β at 48 hpi,with a 11.15 fold change.Meanwhile,NA and BSR cells were used to infect RABV for in vitro experiments,and the results showed that the expression levels of Apo D and ATP5 B in the RABV infected NA and BSR cells could not be significantly affect.The results showed that the transcription and protein expression of Apo D were up-regulated in mouse brain infected,while Apo D transcription was up-regulated in BV2 cells without protein expression.However,there was no significant change in the transcription level of Apo D in NA and BSR cells,indicating that there were great differences in the expression of Apo D protein caused by RABV infection in vivo and in vitro.To further explore the interaction between RABV and Apo D,pc DNA3.0-Apo D eukaryotic expression vector was successfully constructed in this study.On the one hand,RABV first infected BSR cells for 12 h,and then the cells overexpressed Apo D,the results showed that RABV infection could promote the transcription of Apo D.These results indicated that RABV infection could promote the expression of Apo D.On the other hand,BSR cells were transfected with Apo D plasmid for 12 h and then infected with RABV,the results showed that the expression of N and M proteins in the overexpressed Apo D group were increased at 24 hpi and 48 hpi of virus infection.The expression level of N protein of r RC-HL strain was increased 2.86 times and1.89 times,the expression of M protein was up-regulated 2.26 times and 1.61 times,and the expression level of M protein of CVS-11 strain was rise to 1.77 times and 1.95 times respectively.The expression of N protein in r RC-HL△G242-288 strain was increased 3.21 and 1.81 times,and the expression of M protein was up-regulated 1.54 and 1.65 times,respectively.These results indicated that exogenous Apo D could promote the expression of N and M proteins of RABV virus.In summary,RABV infection in mouse brain significantly enhanced the expression of Apo D protein,and virus infection of BV2 cell can enhance the transcription level of Apo D.Meanwhile,it was proved that overexpression of Apo D could promote the expression of N and M protein of RABV virus,but its mechanism remains to be further studied.The data in this study provide theoretical support to explore the interaction between RABV and host proteins Apo D and is helpful to further understand the pathogenic mechanism of RABV.