The Interaction Of Host ?-tubulin And NSP5 Of Group A PoRV G9P[23] Strain And The Effect Of It On PoRV Replication

The group A porcine rotavirus(PoRV)is one of the pathogens causing diarrhea in swines.Non-structural protein 5(NSP5)of PoRV can interact with a variety of viral proteins and play an important role in viral replication.Studies have found that tubulin binds around the viroplasm during the replication of rotavirus(RV).However,the specific mechanism is not yet clear.This study found that NSP5 interacted with ?-tubulin,and studies on many viruses have found that ?-tubulin can interact with viral proteins and thus play a complex role in virus replication.Therefore,it is necessary to explore the effect of ?-tubulin on RV replication.In this study,using the GST pull-down technology coupled with protein mass spectrometry analysis,host cell protein ?-tubulin interacting with NSP5 protein was identified by using prokaryotic expression GST-NSP5 protein as bait protein.In order to further verify the identification results of mass spectrometry,we used the commercialized monoclonal antibody of ?-tubulin and the prokaryotic GST-NSP5 protein as bait protein,verifying the interaction between host cell protein ?-tubulin and NSP5.Furthermore,the interaction of NSP5 protein in eukaryotic cells with ?-tubulin was demonstrated by immunoprecipitation in vitro.And it is proved that NSP5 protein can interact with host cell protein ?-tubulin by the method of Co-IP in the process of group A PoRV/NMTL/G9P[23] strain infection.Moreover,using confocal microscopy and immunofluorescence technology,co-localization of NSP5 protein of group A PoRV/NMTL/G9P[23] strain with host cell protein ?-tubulin in cytoplasm was identified.This study further explored the effect of NSP5 protein interaction with host cell ?-tubulin on the replication of group A PoRV/NMTL/G9P[23] strain.Firstly,using laser confocal microscopy and immunofluorescence technique,?-tubulin depolymerized from the microtubule leading to the depolymerization of microtubule networks in group A PoRV/NMTL/G9P[23] strain.infected MA 104 cells.And,at the same time,the ?-tubulin of microtubule was reduced and granular ?-tubulin appeared in the cytoplasm of virus infected MA 104 cells.In the MA 104 cells treated by nocodazole(inhibition of microtubule polymerization)the ?-tubulin did not exist as a form of granular ?-tubulin,which indicated that granular-tubulin in the process of group A PoRV/NMTL/G9P[23] strain infection is induced by some components of the virus.In addition,this study further explored the effect of host cell ?-tubulin on the replication of group A PoRV/NMTL/G9P[23] strain.In the following transient experiment,it is proved that over expression of ?-tubulin can not only inhibit the synthesis and expression of NSP5 and VP6 proteins of group A PoRV/NMTL/G9P[23] strain,but also inhibit the replication of virus.Through the siRNA interference,it is proved that the reduction of ?-tubulin can not only promote the synthesis and expression of group A PoRV/NMTL/G9P[23] strain NSP5 and VP6 protein,but also promote the replication of virus.By adding nocodazole and ABT-751(?-tubulin in microtubule polymerization inhibition on)that inhibit the polymerization of ?-tubulin from microtubules,not only can promote the expression of NSP5 and VP6 protein,but also promote the replication of the virus;By adding paclitaxel(inhibiting the depolymerization of ?-tubulin on microtubules)proved that inhibiting the depolymerization of ?-tubulin from microtubules not only inhibited the synthesis and expression of NSP5 and VP6 protein,but also inhibited the replication of virus.Therefore,this study suggested that ?-tubulin negatively regulates the replication of the Group A PoRV/NMTL/G9P[23] strain.This study demonstrated the interaction between NSP5 and ?-tubulin for the first time.?-tubulin is the rare host cell protein that can interact with RV NSP5,and it is involved in the replication of group A PoRV/NMTL/G9P[23] strain.The negative regulation of replication of group A PoRV/NMTL/G9P[23] strain suggests that NSP5 may interact with ?-tubulin and affect RV replication.This study will further our understanding of the significance of NSP5 for RV replication and provide new molecular targets for the development of RV vaccines and drugs.