Infectious Bronchitis Virus Attaches To Lipid Rafts And Enters Cells Via Clathrin Mediated Endocytosis

Avian infectious bronchitis is an acute highly exposed respiratory infection caused by avian infectious bronchitis virus(IBV)in chicken.It is characterized by dyspnea,rale of trachea,mouth opening,cough and sneeze.The death rate of nephropathy infection is high and the damage is serious.Infectious bronchitis virus belongs to Coronaviridaeae and Coronavirus-III,which is a single-stranded RNA virus.Up to now,most coronaviruses are known to enter the cells mainly through endocytosis,including SARS-CoV and MERS-CoV,which are harmful to human and animal.It has been reported that the entry of IBV into Baby hamster kidney cells is dependent on the acidic environment,but the specific endocytosis pathway is not clear.In this study,we aim to investigate and characterize the entry pathway of IBV in various cell lines.1.IBV entry is clathrin-mediated endocytosis(CME)dependent.Vero cells,H1299 cells,DF-1 cells,and Huh7 cells were pretreated with CME inhibitor CPZ,CavME inhibitor Nystatin,and Macropinocytosis inhibitor Amiloride,followed with IBV infection.Results showed that CPZ inhibited IBV entry,IBV N protein expression,and progeny virus release(p<0.05),however,Nystatin and Amiloride had no inhibition effect on virus entry and subsequent replication,suggesting that IBV entry requires CME.To further validate the endocytosis pathway exploited by IBV,we knocked down the expression of chathrin heavy chain(CHC)or overexpressed dominate negative mutant of dynamin 1 to block CME,before infecting with IBV.Results showed that IBV entry,IBVN protein expression,and progeny virus release was hampered when CME was specific blocked(p<0.05).These evidences further confirm that IBV enters cells via CME.2.IBV entry is lipid rafts-associated.Cells were pretreated with M?CD to deplete the lipid rafts before infecting with IBV.The disruption of lipid rafts resulted in significant inhibition of IBV entry,subsequent replication and release.(p<0.05).To confirm whether the inhibition effect of the M?CD on virus entry were solely due to cholesterol disruption,we replenished exogenous cholesterol after M?CD treatment,followed with IBV infection.Results showed cholesterol replenishment recovered IBV entry and replication.Immunofluoresence was performed to check whether R18 labeled IBV attaches to lipid rafts.Results showed that R18-IBV bound to plasma membrane and co-localized with lipid rafts marker CM1.These findings revealed that IBV entry is lipid raft-associated.3.IBV entry leads to active actin rearrangement.To examine the role of actin filaments in IBV entry,we used Alexa Fluor 488-phalloidin to staining actin filaments in R18-IBV infected cells at 15 min interval post-infection,and observed the actin filaments arrangement under confocal microscope.We found that with 1-hour post-infection,cells surface displayed significant blebbing,and the number of actin stress fibers dramatically decreased.Meanwhile,R18-IBV moved along with actin filaments.We further investigated the role of the actin cytoskeleton in IBV entry by using inhibitors to block actin polymerization or inhibit depolymerization.We found that IBV internalization was significant increased by inhibitor CytD of Jas treatment in a dose-dependent manner.Above results demonstrate that the IBV attachment and endocytosis lead to actin rearrangement.4.IBV moves across the entire endo-lysosomal system.After identification of the endocytic pathway hijacked by IBV,we tracked the transport vesicles used by incoming IBV.We used the confocal microscope to observe the co-localization between R18-IBV and endosome marker(Rab5: early endosome marker,Rab7: late endosome marker;LAMP1: lysosome marker).We found that IBV moved along with the classical endosome/lysosome track after internalization.To further investigate whether IBV fuses with early endosome or late endosome,we labeled IBV with double dye: R18(red)and DIOC(green).Cells were infected with R18/DIOC labeled IBV,and subjected to immunofluoresence by staining with endosome marker at 1,2 and 3 hours post-infection.Results showed that membrane fusion was induced at 3 h.p.i.(marked with separation of R18 and DIOC signals),the time that virus particles reach late endosomal-lysosomal system.Taken together,our finding demonstrates that IBV attaches to the cell surface lipid rafts,enters the cell via a CME,causes cytoskeleton rearrangement,and moves along with the endo-lysosome system,finally fuses with late-endosome or lysosome at around 3 h.p.i.release viral particles into the cytoplasm and initiate subsequent replication.These results display comprehensive image of IBV entry process and provide antiviral drugs design targets for the prevention and control of IB.