Effect And Mechanism Of Rab5a On Antiviral Immune Response In Respiratory Epithelium With RSV Infection

PART ONE RAB5A IS AN ESSENTIAL HOST PROTEIN FOR RSV REPLICATION IN RESPIRATORY EPITHELIAL CELLSObjective: Respiratory syncytial virus(RSV)is one of the most common viral respiratory pathogen,which causes acute lower respiratory tract infections in infants and young children worldwide,and there is currently no effective vaccine and specific drug treatment.Respiratory epithelium act as the first line of defense against RSV infection.Whether its host protein is involved in resisting viral infection is still unclear.As a vital protein for vesicle transport,Rab family proteins transmit signals within and between cells,participate in immune pathways and inflammatory reactions;In addition,it has been reported that Rab protein is an important host protein involved in the formation of virus replication complex and affects virus replication.Rab protein could affect a series of procedures involving virus life cycle after its entry into the cell.Therefore,this part mainly studies in the role of Rab protein in RSV infection of respiratory epithelium.Methods: We use small interfering RNA to knock down the Rab family protein expression(Rab1a,Rab2 a,Rab4a,Rab5 a,Rab6a,Rab7 a,Rab8a,Rab9 a,Rab11a),and then infect A549 cells with RSV,after that we detect syncytial pathology and detect the copies of RSV N gene,screen out the Rab protein closely related to RSV replication.After clarifying that Rab5 a is an important Rab protein that affects RSV infection,A549 was infected with RSV and cell samples were collected at 1h,2h,6h and12 h after infection.WB and PCR were used to detect Rab5 a protein and gene expression;A549 was pretreated with Rab5 a siRNA and constructed an RSV infection model.The virus adsorption was completed at 4?for 1h,after that the unbound virus particles were washed out,the virus affinity experiment was conducted at 37?.The number of RSV virus particles was counted by immunofluorescence and the expression of RSV N protein in the above processes was determined by co-immunoprecipitation.Virus titer at 12 h,24h,36 h,48h after RSV infection was detected by plaque assays.Construct a plasmid that interferes with the active site of Rab5 a,pretreat A549 cells and then infect the A549 cells with RSV,detect the copies of RSV N gene by PCR.Results: 1.In the RSV-infected A549 model,knockdown of Rab1 a,Rab2a,Rab4 a,Rab5a,Rab6 a,Rab7a,Rab8 a,Rab9a,and Rab11 a affected the formation of syncytia;2.The reduction of Rab4 a,Rab7a,Rab8 a,Rab9a,and Rab11 a protein levels significantly reduced the gene copies of RSV N(P<0.05),but the area of syncytial lesions has no significant difference from that of the control group;The knockdown of Rab5 a protein not only significantly reduced the copies of RSV N gene(P<0.001),but also significantly reduced RSV-induced syncytial pathology;3.RSV infection promoted the gene and protein expression of Rab5 a in A549 in a time-dependent manner,and there was a statistical difference(P<0.05).4.In the model of RSV infection with A549,The number of virus particles in RSV adsorption and affinity process and the expression of RSV N protein were not statistically different between the siRab5 a group and the siCON group.5.Knockdown of Rab5 a significantly reduced the virus titer in A549 after 12h infection(P<0.001).With RSV infection on A549 at 12,24,36,and 48 hours,the viral load of RSV gradually increases with the infection time,at 36 hours The point reached the peak,and the level was still high at48 h.The knockdown of Rab5 a made the viral load of RSV significantly lower than that of the non-knockdown group after 12h(P<0.001);6.After the activation of the site of Rab5 a,RSV N gene copies were significantly increased(P<0.01),While after the inactivation of the site of Rab5 a,the copies were significantly reduced(P<0.01).Conclusion: Rab5 a affects the syncytial pathology,and promotes RSV replication without influencing virus absorption and entry in respiratory epithelial cells,indicating that Rab5 a is an essential host protein in respiratory epithelial cells for RSV infection.PART TWO RAB5 A CONTRIBUTES TO RSV-INDUCED INFLAMMATION AFTER RSV INFECTION OF RESPIRATORY EPITHELIUMObjective: The first part confirmed that Rab5 a is an essential host protein for RSV infection of respiratory epithelial cells,but whether Rab5 a intervene with the virus-induced inflammation in respiratory epithelial cells is not yet clear.Here,we explore the effect of Rab5 a on RSV-induced inflammation in mice models and clinical NPA specimens.Methods: In RSV infected-mice,lung tissue was collected at 12 h,24h,36 h,48h,72 h after infection.WB and PCR were used to detect Rab5 a protein and gene expression,and immunofluorescence to detect Rab5 a expression levels and expression sites;Rab5a shRNA was intranasally given to mice to knock down the level of Rab5 a protein in lung tissue.Plaque assay was used to detect the virus titers of lung tissue at 1h,2h,6h,and 3d after RSV infection,and the total number of inflammatory cells in BALF was counted.HE staining of lung tissue sections of mice after 5d infection and inflammatory pathology was scored.NPA specimens of children,who were admitted to the Lijia Respiratory Department of Children's Hospital of Chongqing Medical University due to acute lower respiratory tract infection caused by RSV infection,were collected during the period from October 2016 to March 2017.The RSV N gene copy numbers and Rab5 a gene expression in the NPA specimens were detected by PCR.IFN-? protein level in NPA specimens were detected by ELISA.and the disease severity of patients was scored according to Wang's score sheet.The correlation among these results was analysed.Results: 1.In the mouse model of RSV infection,Rab5 a was mainly expressed in the bronchial epithelial cells of lung tissue.The gene and protein expression of Rab5 a in the RSV infected group was significantly higher than that in the uninfected group,and reached a peak at 36 h after infection(P<0.01).2.In the mouse model,virus titers of RSV infection at 1h,2h,and 6h were not statistically different between the shRab5 a group and the sh CON group.Besides,the virus titers of the shRab5 a and sh CON groups in lung tissue after BALF and in BALF were not statistically different.3.Knockdown of Rab5 a reduced the virus titer and RSV N gene copy number in lung tissue after 3d infection(P<0.001).4.Rab5 a knockdown significantly reduced the inflammatory cell number and alleviated the inflammation of lung tissue after RSV infection(P<0.01,P<0.001,respectively).5.RSV infection caused increased gene expression of Rab5 a in NPA specimens of children with ALRI(P<0.01),and was significantly positively correlated with the disease severity caused by RSV(P<0.01).6.The Rab5 a expression in NPA specimens was significantly positively correlated with the gene copy number of RSV N(P=0.01).7.The IFN-? level in the RSV infection group in NPA specimens was significantly lower than that in the uninfected group(P<0.001).8.Rab5 a levels and IFN-? levels were significantly negatively correlated in NPA of hospitalized patients with RSV-induced ALRI(P<0.05).Conclusion: RSV promotes the expression of Rab5 a in respiratory epithelial cells and Rab5 a promotes RSV replication,resulting the enhancement of lung inflammation caused by RSV.PART THREE RAB5 A MEDIATES ANTIVRL IMMUNE RESPONSE THROUGH INHIBITING IRF1-DEPENDENT IFN-?Objective: Rab5 a plays an important role in virus infection.It not only participates in the endocytosis of the virus and the formation of microvesicles,but is also closely related to the innate immune response.Interferon is an important factor for virus clearance,including type I IFN and type III IFN.It is reported in the literature that the early endosomes involved in Rab5 a are closely related to the membrane proteins induced by IFN of the IFITM family,and the early endosomes are related to type I interferon receptors,suggesting that Rab5 a is closely related to IFN.In addition,studies have pointed out that IRF1,as an important transcription factor in respiratory epithelial cells against viral infections,is an important regulator of IFN expression,so here we investigate whether Rab5 a regulates IFN expression levels by affecting IRF1,thus mediating the antiviral immune response.Methods: A549 cells were pretreated with Rab5 a siRNA(siRab5a)and its control(siCON)for 24 hours,and then infected with RSV at MOI of 1.The cell supernatant was collected and the levels of IFN-?,IFN-? and IFN-? were detected by ELISA.Cells were collected for PCR to detect the corresponding gene level.Immunofluorescence and WB were used to detect the expression of IRF1 in the nucleus of A549 cells.IRF1 siRNA(si IRF1)was used to pretreat A549,and the culture supernatant was collected to detect the IFN-? level by ELISA.Rab5 a was knocked down in mouse lung tissue with lentivirus that specifically knocks down Rab5a(shRab5a)and its control(sh CON)intranasally,and the mice were infected with 1x10^7 PFU of RSV for 12 and 24 hours,and then lung tissues were collected.The level of interferon in supernatant of lung homogenate was detected by ELISA.WB was used to detect the protein level of IRF-1 in lung tissue,and immunofluorescence was used to detect the expression level of IRF-1 in airway epithelium.After Rab5 a and IRF1 were knocked down by lentivirus(shRab5a)which specifically knocked down Rab5 a and IRF1,the lung tissues were collected.WB was used to detect the level of IRF1 in mouse lung tissue,and the level of IFN-? in the supernatant of mouse lung homogenate was detected by ELISA.Results: 1.After RSV infection with A549,IFN-?,IFN-?,and IFN-? protein and gene levels were significantly increased(P<0.001).After Rab5a was knocked down,IFN-? and IFN-? protein and gene levels were slightly increased,while the IFN-? protein and gene levels were significantly increased(P<0.01).2.The IFN-? level of lung tissue homogenization in RSV infection group at 12 h and 24 was significantly reduced(P<0.001).After Rab5 a was knocked down,the IFN-? level was significantly higher than that in the sh CON group P<0.001).3.In RSV-infected A549,the expression of IRF1 in the nucleus was increased(P<0.001),knocking down Rab5 a further promotes IRF1 into the nucleus 4.In RSV-infected A549,the IFN-? level in the IRF1 knockdown group was significantly lower than that of the control group(P<0.01).The concentration of IFN-? after Rab5 a and IRF1 knockdown at the same time was higher than that of the IRF1 knockdown group alone.There was a statistical difference(P<0.01).5.After RSV infection,the expression of IRF1 in the lung tissue increased.After Rab5 a was knocked down,the level of IRF1 was significantly increased(P<0.05),and the expression of IRF1 in the nucleus increased(P<0.01).Rab5 a knockdown promoted the translocation of IRF1 into nucleus(P<0.01).6.In the mouse model of RSV infection,the level of IFN-? in the lung homogenate of mice was significantly lower than that in the uninfected group;knocking down Rab5 a significantly increased the level of IFN-?(P<0.001),knocking down Rab5 a while knocking down IRF1 expression at the same time,IFN-? level was significantly lower than that in the Rab5 a knockdown group alone(P<0.01).Conclusion: In RSV-infected respiratory epithelial cells,the expression of Rab5 a increases,thus inhibiting the antiviral immune response of epithelial cells by regulating IRF1 to down-regulate the level of IFN-?.