| Rabies is a highly fatal zoonosis caused by rabies virus(RABV)infection,RABV caused acute and fatal nervous system infection in all warm-blooded animals,including humans.RABV is transmitted through bites or scratches by rabies animals.The virus spreads from the infected site to the central nervous system(CNS)along neurons,where the virus replication causes transmission and symptom occurrence.Once appeared clinical symptoms,the mortality rate is almost 100%.However,because it is not completely clear about the pathogenesis,so that there is no effective clinical treatment.Interferon-induced transmembrane protein 3(IFITM3)is an important host effector molecule of type I interferon in response to virus infection.Relevant reports have confirmed that interferon(IFN)signal pathway plays an important role in the innate immune response during RABV infection and IFITM3 is an interferon induced protein.While the role of IFITM3 in RABV infection has not been clarified.Therefore,this study systematically studied the mechanism of IFITM3 in RABV infection.In this study,we found IFITM3 not only inhibits the replication of RABV infection through autophagy but also proved that IFITM3 plays an indispensable role in the response to interferon against RABV.The exogenous expression of IFITM3 significantly inhibits the infection of RABV,while knockdown IFITM3 has the opposite effect.Then we found that IFNβ up-regulated IFITM3,and IFITM3 can regulate the IFNβ triggered by RABV in a positive feedback way.We further found that IFITM3 not only inhibits virus adsorption and entry,but also inhibits virus replication through m TORC1-dependent autophagy.These findings provide a direction for systematically clarifying the role and molecular mechanism of IFITM3 in RABV infection and provide potential drug targets for the treatment of rabies.(1)IFITM3 expression in different cells after infected RABVRT-qPCR detects the basal level of IFITM3 was different in a variety of different cells.The results showed that the cells with the basal level of IFITM3 from low to high were diverse: MDCK,BHK,HEK-293,SVGP12,293 AD,N2 a,MA-C,A549.Western blot was used to detect the cell samples of A549 cells after infected RABV 24,48,72 hours.It was found that as the infection time increased,the expression of IFITM3 was increased,while the replication of RABV was decreased;but the opposite situation occurred in 293 AD cells.Immunofluorescence detected that IFITM3 changed from a diffuse distribution to a dot distribution after infected RABV,and colocalized with RABV.(2)IFITM3 inhibits the infection and replication of RABVDetect the viral replication at multiple time points in MDCK overexpression cell line and A549 cell line through indirect immunofluorescence,Western blot and viral load.used 0.1 MOI and 1 MOI CVS-11 infected cells respectively.Used 0.1 MOI and 1 MOI CVS-11 infected two types of cells.Through Western blot detect the cell samples that infected CVS-11 after 12 and 24 hours.It was found that overexpression IFITM3 significantly decreased viral infection and replication,while silencing of IFITM3 significantly enhanced viral infection and replication.Immunofluorescence and viral load detection showed the same results.Because the overexpression of IFITM3 in MDCK cell line needs to be induced by DOX,wetreated MDCK cells with DOX and found that DOX did not inhibit the infection and replicationof RABV.(3)IFITM3 inhibits virus adsorption and entryTo determine whether IFITM3 affects the adsorption or entry of RABV,used q RT-PCR and Western blot to detect the adsorption and entry of RABV in IFITM3 silenced cell line and overexpression cell line.Compared with the negative control,when IFITM3 was silenced,the virus copy increased moderately but not significantly.It is believed to be caused by the promotion of virus replication induced by silent IFITM3.At the same time,the overexpression of IFITM3 in 293 AD cell line effectively reduced the virus copy.Consistent data were observed in the Western blot results.These results showed that the overexpression of IFITM3 inhibited the adsorption and entry of RABV at m RNA and protein level,while IFITM3 silencing did not reduce the adsorption and entry of the virus,which may be caused by the compensation of receptor diversity.(4)IFITM3 inhibits RABV replication by inhibiting m TORC1/ULK1 dependent autophagyIn A549 cells,related protein of autophagy was detected after silenced IFITM3 by Western blot.We found that RABV infection promoted autophagy,meanwhile,silence IFITM3 promoted autophagy and virus replication.In 293 AD cell line,overexpression IFITM3 inhibited autophagy that caused by viral infection.However,whether IFITM3 was silenced or overexpressed,the expression of P62 protein is basically consistent.It is speculated that RABV does not induce complete autophagy,Subsequent experiments were tested using the LC3 dual fluorescence lentivirus system.The A549 cell samples that handle with transfection and multi-time point exposure were detected by LC3 double-fluorescence lentivirus system,and the changes of multitime point autophagy flow were observed under fluorescence microscope.It was found that silencing IFITM3 promoted the accumulation of cytoplasmic autophagy(RFP-GFP)whether infected RABV.However,in the case of the overexpression of IFITM3,RABV-induced autophagy decreased.No obvious autolysis was observed until 24 hours.Because GFP fluorescence decreased after lysosomal acidification(p H value decreased),while RFP fluorescence remained stable.These results suggest that IFITM3 does play an anti-RABV role by inhibiting m TORC1/ULK1-dependent incomplete autophagy.(5)IFITM3 up-regulate IRF7 in protein level,and IFITM3 positive regulates the expression of IFNβ after infected RABVUsed RT-qPCR found that IRF7 was significantly upregulated at mRNA level after infected RABV,and IRF7 also belonged to the upstream regulatory factor of IFITM3.Meanwhile,some studies have found that the insensitivity of virus in cells is related to the expression level of IRF7 and interferon related genes,thus mediating the antiviral response of cells.Western blot analysis of cell samples after silencing IFITM3 and IRF7 showed that the expression of IRF7 was downregulated after silencing IFITM3,and the replication of virus infection was enhanced;However,after silencing IRF7,the expression level of IFITM3 is not regulated,and virus infection replication is also enhanced.Because IFNβ is an upstream regulatory factor of IFITM3,and we examine whether and how IFITM3 effects the IFNβ triggered by RABV.ELISA and Western blot showed that after IFITM3 silenced A549 cells were infected with RABV,the protein level of IFNβ was decreased.Indicates that silencing IFITM3 inhibits the protein level of IFNβ which induced by RABV.The experimental results indicate that IFITM3 positively regulates IFNβ after infection RABV.(6)IFNβ promote the expression of IFITM3To determine whether IFITM3 added by RABV is IFNβ dependent,used the restructured person IFNβ Protein treatment the A549 cell line that infected RABV.The virus replication was significantly reduced,and the expression of IFITM3 was significantly increased whether infected RABV or not.When we use si RNA blocked-out IFNβ in A549 cell line and then infected RABV,the expression of IFITM3 was inhibited and the replication of the virus was enhanced,indicating that IFITM3 induced by RABV was IFNβ Dependent.Then we determine whether IFNβ is the main effector mediates IFITM3 to anti-RABV,but IFNβ still anti-RABV in A549 cell line which was knocked-out IFITM3,indicating that IFITM3 is not the only downstream effector of IFNβ in anti-RABV,although IFITM3 is controlled by IFNβ.(7)IFITM3 protein expression is not regulated by miRNAs,but is degraded by protease systemMicro RNA(miRNA)has many important regulatory roles in cells.It is a class of non-coding single-stranded RNA molecules encoded by endogenous genes,with a length of about 20-24 nucleotides.miRNA often regulates target m RNA by destroying its stability and inhibiting its translation.We predicted IFITM3-related miRNAs by software,then detected the expression of related miRNAs in multi-time points.The expression of hsa-miR-494-3p、hsa-miR-142-5p and hsa-miR-15b-5p in m RNA level decreased with the increase of RABV infection time.Meanwhile,transcription factor testing revealed that hsa-miR-486-3p was downregulated after infected RABV.Western blot detected that there was no change in the expression level of IFITM3 after overexpression and silencing of hsa-miR-486-3p,and overexpression and silencing of hsa-miR-15b-5p did not affect the replication and infection of RABV.To further explore whether the upregulation of IFITM3 in A549 induced by RABV was affected by the degradation of protein after exposure,we measured the half-life of IFITM3 by using cycloheximide(CHX)to detect the stability of IFITM3 protein after RABV infection.RABV infection increased the half-life of IFITM3 from 13.64 hours to 29.36 hours,indicating that the upregulation of IFITM3 induced not only by IFNβ,but may also be affected by the reduction of IFITM3 degradation.In summary,this study was the first found that IFITM3 inhibited the replication of RABV infection in cells.It was also found that IFNβ induce a significant upregulation of IFITM3,which has an inhibitory effect on the virus.IFITM3 also could inhibit viral replication by inhibiting the occurrence of autophagy.The antiviral effect of IFITM3 provides a theoretical basis for anti RABV target. |
