Research On The Function And Mechanism Of Exo70 Gene In The Replication Of Bovine Hepersvirus ?

Bovine herpes virus I(BoHV-1),also known as infectious bovine rhinotracheitis virus(IBRV),belongs to the subfamily of ?-herpesvirus.BoHV-1 can cause acute,febrile and contagious IBR,which occurs and is prevalent to varying degrees in many countries and regions in the world.In the developed countries of cattle industry,the prevention and control of IBR is mainly based on based on vaccine immunization and combined with the strategy of purifying continuously infected cattle.Although the inactivated single vaccine of IBR has been developed in China,it has not been widely applied.At the same time,vaccine immunization can not eliminate persistent infection.Therefore,while focusing on vaccine immunoprophylaxis and the development of new vaccines,it is necessary to further study the mechanism of virus replication,especially the molecular mechanism of virus assembly and release,in order to find the key genes regulating the replication of BoHV-1 and provide target molecules for the development of antiviral drugs.Exo70 protein is a key subunit among the eight subunits of exocyst complex,which is involved in intracellular transport of secretory vesicles,mediates the anchoring of secretory vesicles on the plasma membrane,and promotes the excretion of substances in secretory vesicles.Although Exo70 plays an important role in maintaining cell morphology,building cell connections and promoting cell migration,the relationship between Exo70 and virus replication has been rarely reported.The secondary envelope of virus particles and the formation of virus secretory vesicles are completed on the Trans Golgi network(TGN),but the mechanism of transport from the Golgi to the plasma membrane and its release is still poorly understood.In this study,the molecular mechanism of Exo70 promoting the replication of BoHV-1 was discussed based on the analysis of the relationship between Exo70 and the transport and release of virus secretory vesicles to the plasma membrane.The main experimental results are as follows:(1)Exo70 promotes the release of BoHV-1.The expression of Exo70 in MDBK cells infected by BoHV-1 at different time points was detected by qPCR and Western blot.The results showed that Exo70 mRNA level was increased in MDBK cells infected with BoHV-1,and the expression of Exo70 protein level was up-regulated 12 h,but down-regulated 24 h after virus infection.After the overexpression of Exo70 gene,compared with the control group,the titer of the virus in the supernatant increased from 106.4 TCID50/mL to 107.07 TCID50/mL,increased by 100.67 times,but the total titer unchanged.After using shRNA to suppress the expression of Exo70,the virus titer in the supernatant decreased from 106.93 TCID50/mL to 105.8 TCID50/mL,decreased by 101.13 times,the total titer remained unchanged.These results indicate that Exo70 does not affect the replication of the virus,but affects the release of the BoHV-1.(2)Exo70 facilitates the transport of BoHV-1 secretory vesicles to the plasma membrane.Transmission electron microscopy was used to observe the samples of MDBK cells infected by BoHV-1.It was found that the secondary envelope of BoHV-1 appeared on TGN.After blocking the Golgi-plasma membrane transport pathway with BFA and Monensin treatment,immunofluorescence technology was used to detect the MDBK cells infected by BoHV-1.In the control group of non drug treatment,the co-localization of BoHV-1 gB protein and GM130(Golgi marker)was less,and it was distributed in cytoplasm and plasma membrane.After BFA treatment,GM130 presented a diffuse distribution and the co-located between GM130 and gB protein was obvious,and gB on the plasma membrane was significantly reduced.Cells treated by Monensin,GM130 presents the state of aggregation,the co-localization of gB protein and GM130 increased significantly,and gB was mainly distributed in Golgi body,which was not detected on plasma membrane.Through transmission electron microscopy,the monensin-treated group had more virions near the Golgi body than the control group,while the number of virions released to the extracellular was significantly less than the control group.The results showed that the transport of BoHV-1 to the plasma membrane depends on the Golgi-plasma membrane transport pathway.The co-localization of Exo70 with gB protein during BoHV-1 infection was verified by immunofluorescence labeling.After inhibiting the expression of Exo70,BoHV-1 gB protein was found to be mainly concentrated and distributed in the cytoplasm,co-located with GM130,and rarely distributed on the plasma membrane.Transmission electron microscopy showed that there were more virus particles in the cells after the inhibition of Exo70 expression than in the control group.However,after inhibiting the expression of Exo70,the number of virions released to the extracellular was significantly lower than the control group.The above results indicated that Exo70 facilitates the transport of BoHV-1 to the plasma membrane.(3)Rablla may be involved in the intracellular transport of BoHV-1 regulated by Exo70.qPCR and Western Blot were used to detect BoHV-1 infected cell samples.Compared with control cells,Rablla mRNA expression was always up-regulated;the expression of Rablla protein was up-regulated 12 h,but down-regulated 24 h after infection.TCID50 examined the effects of Rablla on BoHV-1 replication,and found that Rablla promoted BoHV-1 replication.After inhibits the expression of Rablla by shRNA,the titer of the virus decreased from 107.42 TCID50/mL to 106.76 TCID50/mL,decreased by 100.66 times.Immunofluorescence technique was used to detect the co-location of virus gB and Rablla,and BoHV-1 infection promoted the cytoplasmic localization of Rab11a and Exo70.Immunocoprecipitation technique was used to detect the interaction between Rab11a and BoHV-1 gD,gE,and the binding of Rablla to Sec6 Sec10,the subunit of exocyst complex,and the binding of Rablla to Sec10 were promoted by the infection of BoHV-1.Immunofluorescence technology was used to locate the gB protein.It was found that the inhibition of Rablla inhibited the transport of BoHV-1 to the plasma membrane.The above results showed that Rablla may be involved in the Exo70 regulated intracellular transport of BoHV-1(4)At the later stage of BoHV-1 infection,the E3 ubiquitin ligase STUB1 promoted the ubiquitination of Exo70.At the late stage of viral infection,Exo70 protein expression was down-regulated,and the expression of Exo70 protein was reversed after MG 132 treatment,indicating that Exo70 protein had ubiquitination degradation.Through affinity purification and mass spectrometry analysis,high-throughput screening of Exo70 binding proteins,STUB1 was found to be a candidate E3 ubiquitin ligase;the co-localization of STUB1 and Exo70 during BoHV-1 infection was detected by immunofluorescence labeling;the interaction between STUB1 and Exo70 was determined by co-immunoprecipitation.Co-transfected the recombinant expression plasmids of STUB1 and Exo70,found that STUB1 promoted the ubiquitin modification of Exo70,inhibited the expression of Exo70 protein,and the overexpression of STUB1 shortened the half-life of Exo70.After mutating the enzyme activity site of STUB1,immunoprecipitation assay revealed that the ubiquitination of Exo70 by STUB1 was dependent on its enzyme activity.These results indicate that host cells promote the ubiquitination degradation of Exo70 by E3 ubiquitin ligase STUB1 at 24 h after viral infection.To sum up,this study first discovered the mechanism of Exo70 gene promoting the intracellular transport and release in the process of BoHV-1 replication,which provides a new perspective and platform for in-depth study of the replication mechanism of BoHV-1,provides direct evidence for understanding the role and mechanism of Exo70 in the life cycle of virus particles,and provides new ideas and target molecules for the development of anti-BoHV-1 drugs.