The Pathway Of Newcastle Disease Virus Entering Dendritic Cells And DF1 Cells

Newcastle disease,which causes respiratory tract and the digestive tract damage,is a highly contagious and fatal viral disease.It is caused by Newcastle disease virus(NDV)which belonging to the family Paramyxoviridae,subfamily Avulavrius with the single stranded,non-segmented,negative genomic RNA virus.Privious studies have begun to reveal the mechanism of NDV invading host cells.In the case of paramyxoviruses,including NDV,it has been established that viruses can utilize the membrane fusion process and endocytosis pathways to enter cells.Besides,other reports indicated that NDV may infect He La cells through a caveolae-dependent endocytic pathway.However,virus can use different strategies to entering different cells.In this study,we used different methods such as pharmacological inhibitors and si RNA interference to study the pathway of NDV entering DF1 cells and DCs.1.The function of Clathrin,dynamin and cholesterol in NDV entering and infecting cellsZJ1 was used to infect DF1 cells and DCs,results revealed that NDV completed the replicative cycle within 4h.We used CPZ,dynasore and M?CD in appropriate concentration to pretreat cells,and the results indicated that CPZ and dynasore can inhibit the expression of NP significantly(p<0.05),but M?CD can not.To further evaluate the role of clathrin on NDV internalization,its expression in DF1 cells was knocked down using CHC-specific si RNAs and confirmed by Western Blot,NDV internalization was greatly inhibited after CHC knockdown.These findings suggested that entry of NDV into DF1 cells and DCs is dependent on CME and dynamin but not dependent on cholesterol.2.The function of macropinocytosis in virus enteringWe choosed Cyto D to inhibit actin rearrangement,Jasplakinolide to stabilize actin rearrangement,and EIPA to inhibit NHEs.The results indicated that both pharmacological inhibitors can inhibit the expression of NP in cells,so entry of NDV need actin rearrangement and NHEs.When cells were pre-treated with wortmannin,a inhibitor of PI3 K,we found no inhibition of NDV entering DF1 cells while decrease in DCs.To further examine the role of PKC,NDV-infected cells were pre-treated with Rottlerin and PMA,respectively.Results revealed that Rottlerin effectively inhibited NDV internalization.By contrast,NDV entry increased in PMA-treated cells.We then detected changes in the levels of activated Rac1 and p-PAK at indicated times during NDV early entry.The results showed a slight increase in activated Rac1 and p-PAK and the trend is nearly the same.Meanwhile,pretreating cells with a Rac1 inhibitor greatly decreased NDV entry.In sum,the Rac1-Pak1 pathway is involved in NDV entry,besides,NDV can use macropinocytosis to enter cells.3.Intracellular trafficking of NDV in cellsWe transfected DF1 cells with Rab5a-wt,Rab5a-S34 N,Rab7-wt,Rab7-T22 N for 24 h and then to detect intracellular trafficking of NDV.The results indicated that overexpression of wild-type Rab5a(Rab5wt)significantly increased NDV infection,whereas overexpression a DN Rab5 mutant(Rab5-S34N)significantly inhibited NP expression.The overexpression of Rab7-T22 N and Rab7 wt in DF-1 cells had no effect on NDV infection.In addition,EEA1 can co-localize with NP while LAMP1 can not.This indicates that NDV peep off capsid in early endosome.In conclusion,NDV can utilize clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter cells.This findings shed new light on the molecular mechanisms underlying NDV entry into cells,and provide new thoughts and methods for protecting ND.