Interaction Between Host Rab22a And CSFV NS4B Protein And Its Effect On CSFV Replication And Proliferation

Classical swine fever(CSF),which caused by classical swine fever virus(CSFV),is a infectious diseases of pigs,and very harmful to the pig industry.In eukaryotic cells,materials in different organelle are transferred through vesicle transport pathway.It has been found that Ras-related gtp-binding protein(Rab)participate in vesicle transporting and with regulating function,which playing a crucial role during vesicle transporting.The viral activities are correlative to multiple Rab which include Rab22 a.Rab22a,a member of the Rab family small GTPases widely express in mammalian cells.In the research of our team previously,there is possibility of interaction between Rab22 a and NS4 B protein of CSFV which was proved by yeast two-hybrid system.Therefore,the work of exploring the interaction between NS4 B protein of CSFV and Rab22 a and researching the regulatory effect of Rab22 a on CSFV replication and proliferation were performed in this study.The results obtained from this research are as follows:(1)Rab22a interacts with CSFV NS4 B.First,we catch sight of endogenous Rab22 a interacted with CSFV NS4 B by Co-IP assay in PK-15 cells;next,the direct interaction mode between Rab22 a and CSFV NS4 B is demonstrated by GST pull-down assay;finally,the Laser Scanning Confocal Microscope(LSCM)picture shown that Rab22 a and CSFV NS4 B co-localized in PK-15 cells.(2)Rab22a promotes the replication of CSFV.Post transfection with Rab22 a overexpression vector into PK-15,there are significant up-regulation of CSFV RNA that detected by RT-q PCR,which was extremely significant up-regulation at 24(P<0.01)and significant up-regulation at 48 h and 72 h(P<0.05),there are the same variation tendency of the CSFV E2 protein expression all the time(P<0.01).However,the replication of CSFV was blunted when Rab22 a was interfered with RNA interference technique except the point of 24 h(P>0.05),the expression of CSFV RNA were significant inhibited at 48 h(P < 0.05)and extremely significant inhibited at 72 h(P<0.01);meanwhile,the expression of CSFV E2 protein were extremely significant inhibited at 48 h and 72 h(P<0.01,P<0.001).In addition,we introduced two point mutation vector,Rab22 a Q64L and Rab22 a S19N,successfully,which influence the replication of CSFV post transfection into cells,the expression of CSFV RNA and CSFV E2 protein were significant inhibited at 24 h(P<0.05);Rab22a Q64 L significantly inhibited them(P < 0.05)and Rab22 a S19N had extremely significantly inhibition effect on them at 48 h(P<0.01),while at the point of 72 h this two mutants both have the extremely significant inhibition effect(P<0.01).(3)Rab22a can promote the proliferation of CSFV.The results are that over-expression of Rab22 a can significant promote the production of CSFV's progeny virions(P<0.05);on the contrary,the interference of Rab22 a significant inhibited the production of CSFV's progeny virions(P < 0.05);the production of CSFV's progeny virions was significantly inhibited by the expression of Rab22 a Q64L and Rab22 a S19N at 24 h(P<0.05),and was extremely significant inhibited at 48 h and 72 h(P<0.01,P<0.001).In summary,this study shows that Rab22 a is one of host protein which interacts with CSFV,and promoting CSFV intracellular proliferation.This research shed light on the relationship between host Rab22 a and CSFV and having accumulated novel scientific data.