| Peste des petits ruminants(PPR)is a contagious disease caused by the peste des petits ruminants virus(PPRV).It mainly infects small ruminants such as sheep and goats and can also infect wild ungulates such as antelope and deer.PPRV infection can cause symptoms including fever,pneumonia,diarrhea,and abortion in pregnant ewes.Its incidence in goats is30%,and the mortality rate can be as high as 90%.PPRV mainly infects lymphocytes and epithelial cells of infected hosts,leading to immunosuppression and increased susceptibility to secondary infection.Interferon-induced transmembrane proteins(IFITM)are a family of small transmembrane proteins induced by interferons(IFNs).They are mostly localized on the early endosome,late endosome,and lysosome membranes and play a key role in regulating virus entry and replication in the early stage of viral infection.Currently,the endosomal transport pathway of PPRV in host cells and the role of IFITM in the early stage of PPRV infection are still unclear.The purpose of this study was to detect the types of endosomes involved in PPRV infection and the effects of PPRV infection on the expression of endosomerelated molecules(IFITM1,IFITM3,Rab5,and Rab7)in goat endometrial epithelial cells(EECs)after PPRV infection.The study aimed to explore the effect of IFITM1 and IFITM3 on viral replication in the early stage of PPRV infection and its mechanism.The main contents and results are as follows:1.To determine the types of endosomes involved in the intracellular transport of PPRV in goat EECs,the co-localization of PPRV N protein and EEA1(an early endosome marker molecule),Rab7(a late endosome marker molecule),and LAMP1(a lysosome marker)in PPRV-infected EECs(MOI=5)was detected by immunofluorescence.The results showed that PPRV N protein co-localized with EEA1,Rab7,and LAMP1,indicating the involvement of both early and late endosomes in the early endosomal transport of PPRV infection.To further confirm the effect of PPRV infection on the expression levels of IFITM1,IFITM3,Rab5,and Rab7 in goat EECs,the expression levels of IFITM1,IFITM3,Rab5,and Rab7 were detected by Western blotting within 6 hours after PPRV(MOI=5)infection of goat EECs.The results showed that compared to the uninfected control group,PPRV infection significantly enhanced the expression levels of IFITM1,IFITM3,Rab5,and Rab7 in goat EECs in the early stage of infection,and this enhancement was time-dependent.2.To investigate the role of IFITM1,IFITM3,Rab5,and Rab7 in the early stage of PPRV infection,goat EECs were transfected with specific small interfering RNA(siRNA)(5 n M)or overexpression plasmids(1.5 μg/m L)targeting IFITM1,IFITM3,Rab5,and Rab7,along with their corresponding controls.Subsequently,the cells were inoculated with PPRV(MOI=5).The replication and proliferation of PPRV were assessed by immunoblotting and TCID50 assays at 2 hours post-infection.The results indicated that compared to the negative control group,the knockdown of IFITM1,IFITM3,Rab5,or Rab7 significantly inhibited the expression of the N protein in the early stage of PPRV infection and reduced the virus titer in the cell culture supernatant.Conversely,the overexpression of IFITM1,IFITM3,Rab5,or Rab7 promoted the expression of the N protein in the early stage of PPRV infection and increased the virus titer in the cell culture supernatant.These findings demonstrate that IFITM1,IFITM3,Rab5,or Rab7 can significantly enhance the proliferation level of PPRV in goat EECs.3.To determine the effect of PPRV infection on interferon production in goat EECs,the transcription level of IFN-β was assessed by q RT-PCR within 6 hours after PPRV(MOI=5)infection of EECs.The results revealed that compared to the uninfected control group,PPRV infection significantly inhibited the transcription level of IFN-β in goat EECs at the early stage.To further investigate the role of IFITM1 and IFITM3 in regulating the transcription level of IFN-β during PPRV infection,different doses of siRNA targeting IFITM1 and IFITM3(50n M,100 n M,150 n M)or overexpression plasmids(1 μg/m L,1.5 μg/m L,2 μg/m L)and their corresponding controls were transfected into goat EECs.After 24 hours,PPRV(MOI=5)was inoculated,and the transcription level of IFN-β was measured 2 hours after infection.The results demonstrated that compared to the negative control group,the knockdown of IFITM1 and IFITM3 significantly promoted the transcription level of IFN-β,while the overexpression of IFITM1 and IFITM3 inhibited the transcription level of IFN-β.4.To investigate the mechanism by which IFITM1 and IFITM3 regulate the transcription level of IFN-β during PPRV infection,siRNAs targeting IFITM1 and IFITM3(100 n M)and their corresponding controls were transfected into goat EECs.After 24 hours,PPRV(MOI=5)was inoculated,and the expression levels of IRF3 and phosphorylated IRF3 were assessed by immunoblotting at 2 hours post-infection.The results demonstrated that the knockdown of IFITM1 and IFITM3 significantly accelerated IRF3 phosphorylation compared to the negative control group,indicating that IFITM1 and IFITM3 can inhibit IRF3 phosphorylation.To further explore the mechanism by which IFITM1 and IFITM3 inhibit IRF3 phosphorylation,different doses of siRNA targeting IFITM1 and IFITM3(50 n M,100 n M,150 n M)or overexpression plasmids(1 μg/m L,1.5 μg/m L,2 μg/m L)and their corresponding controls were transfected into goat EECs.After 24 hours,PPRV(MOI=5)was inoculated,and the expression levels of phosphorylated IRF3,LC3-I,and LC3-II were detected by immunoblotting at 2 hours post-infection.The results revealed that compared to the negative control group,the knockdown of IFITM1 and IFITM3 significantly inhibited LC3-II expression and promoted the phosphorylation of IRF3,while the overexpression of IFITM1 and IFITM3 significantly promoted LC3-II expression and inhibited the phosphorylation of IRF3.These findings suggest that IFITM1 and IFITM3 inhibit IFN-β transcription by inducing autophagy to degrade the levels of phosphorylated IRF3.5.To examine the effect of different PPRV proteins on the expression of IFITM1 and IFITM3,plasmids encoding PPRV N,H,M,F,C,and V genes(1.5 μg/m L)were transfected into goat EECs,and the expression levels of IFITM1 and IFITM3 were assessed by immunoblotting after 24 hours.The results demonstrated that compared to the negative control group,the expression of IFITM1 and IFITM3 increased following the transfection of C,M,and F gene plasmids.These findings suggest that PPRV C protein,M protein,and F protein play an important role in inducing the expression of IFITM1 and IFITM3 during PPRV infection.In summary,this study revealed that during the early stage of PPRV infection,the sorting and transportation of early and late endosomes in host cells are crucial.The infection of PPRV induces the expression of IFITM1 and IFITM3,as well as the endosomal transport regulatory proteins Rab5 and Rab7,which play a significant role in promoting virus proliferation.Furthermore,the study elucidated the role and mechanism of IFITM1 and IFITM3 in inhibiting the transcription of IFN-β during the early stages of PPRV infection in sheep epithelial cells. |
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