Study Of The Relationship Between Bif-1 Protein And Rabies Virus Replication

Rabies,also known as hydrophobia,is a zoonotic diseasethat is caused by infection with viruses of the Lyssavirus genus.Rabies virus?RABV?,the prototype virus of the Lyssavirus genus,is the most common causative agent of rabies by now.Usingvaccines and following reasonable programs can protect against the infection by RABV.However,it is almost 100%lethality once the clinical sympotom occurred,due to the lack of effective therapeutic methods.China is one of the countries with high incidence of rabies,ranking second all over the world.Therefore,promoting the study of pathogenic mechanism of RABV and finding the curative modifiers are urgent and necessary.Autophagy is a vital important intracellular catabolic process for aberrant cytoplasmic materials both endogenous and exogenous.Piled up evidences showed that autophagy was involved in the progress of pathology ofviruses.Vesicular stomatitis virus?VSV?induced autophagy is a kind of innate immunity response and inhibits the replication of VSV,while influenza A virus replication inhibits the induction of autophagy to benefit its infection.However,there are few reports about RABV induced autophagy.RABV challenge virus standard?CVS?-11is a virulent strain that can cause severe clinical symptom and final death in mice.The result of previous study by proteomicsanalysis in our lab showed that the differently expressed proteins in RABV infected mouse brains are highly targeting on autophagy pathway.Bax-interacting factor-1?Bif-1?,a highly expressed protein in RABV infected mouse brains,was selected as the object of this study.The relationship between the expression of Bif-1 in host and the replication of RABV?CVS?-11 strain,the relationship between Bif-1 and RABV induced autophagy and also the relationship between Bif-1 and RABV induced apoptosis were tentatively explored by using several molecular biological approaches in vitro.Chapter 1:The influence of RABV replication on the expression of Bif-1.NA cells and BHK cells were infected with RABV CVS-11 strain respectively.The samples of total RNA and total proteins were harvested per 12h.Meanwhile,uninfected cellswere serviced as control.The expression of Bif-1 was detected by real-time quantitative PCR?RT-qPCR?in mRNA level and by western blot?WB?in protein level.The results show that the expression of Bif-1 mRNA was elevated all time but the expression of Bif-1protein only rose up in early stage and declined in later in RABV infected NA cells;the expression of Bif-1 both in mRNA level and protein level decreased in RABV infected BHK cells.The study in this part suggested there were diferencies for the expression of Bif-1 in RABV infected NA cells and BHK cells.Chapter 2:The influence of Bif-1 gene silencing on RABV replication.The plasmids expressing Bif-1 targetting shRNA were designed and screened for validity.The plasmids were transfected in NA cells and BHK cells using liposome reagent for24h.Meanwhile,cells in control group were transfected with the plasmids expressing nontargeting shRNA.Then cells were inoculated with RABV CVS-11 strain,and the total RNA and the cell culture supernatant were harvested at 24 hpi and 48 hpi.RT-qPCR was performed to detect the production of virus genomic RNA and TCID₅₀was performed to detect the production of progeny viuses.The results show that Bif-1gene silencing promotedRABV replication in NA cells but inhibited RABV replication in BHK cells.The study in this part suggested that knockdown of Bif-1 in NA cells and BHK cells has different influence on RABV replication.Chapter 3:The influence of Bif-1 gene overexpression on RABV replication.Eukaryotic expression plasmids of 7 Bif-1 alternatively spliced isoforms of mus musculus were constructed.Methods of transfection,inoculation and detection were the same as chapter 2.Meanwhile,cells in control group were transfected with empty plasmids.The results show that the overexpression of Bif-1b,Bif-1c and Bif-1e isforms inhibited RABV replication in NA cells,while the overexpression of Bif-1a,Bif-1b,Bif-1c andBif-1e isforms promoted RABV replication in BHK cells.The study in this part suggested that overexpression of different Bif-1 isoforms in NA cells and BHK cells has different influence on RABV replication.Chapter 4:The influence of Bif-1 gene expression on RABV induced autophagy and apoptosis.Normal and Bif-1 overexpressed NA cells were inoculated with RABVCVS-11 strain.Electronic microscopy was performed to detect the generation of autophagosomes.Confocal Laser Scanning Microscopy was performed to detect the colocalisation of Bif-1 and LC3.Flow cytometry?FCM?was performed to detect the apoptosis induced by RABV.WB was performed to detect the protein level changes of autophagic and apoptotic markers.The results show that RABV induced incompleted autophagy companied with apoptosis in NA cells;Bif-1 colocalized with LC3 after RABV infection;overexpression of Bif-1 enhanced the blocked autophagic flux and resisted apoptosis induced by RABV.In conclusion,this study illustrated the relationship between Bif-1 and RABV replication from host protein expression and virus infection points,and tentatively explored the function of Bif-1 in RABV induced autophagy and apoptosis in NA cells,suggesting Bif-1c may be a promising target for discovering new anti-RABV drugs.