Effects Of Mouse DDX18 On IFN-? Production And Virus Proliferation

DEAD-box family proteins are a large number of highly conserved RNA helicases,which are involved in various biological processes such as mRNA transcription and nuclear export,pre-RNA splicing,RNA binding and hydrolyzing,small RNA synthesis and protein translation.In recent years,a large number of studies have found that DEAD-box RNA helicases are also involved in innate immune regulation,especially in regulating the production of interferon,however,different DEAD-box RNA helicases have differrent effect on the regulation of IFN production and downstreamsignaling pathways.Previous studies in our laboratory found that porcine-derived DDX18?p DDX18?and human-derived DDX18?h DDX18?interact with the transcription factor IRF3 and inhibit IRF3-mediated activation of IFN-? promoter,thereby inhibiting IFN-? production,which confirmed that DDX18 functions in regulating interferon at cellular level.To further confirmthe effect of DDX18 on the regulation of interferon in vivo,our study firstly verified that mouse-derived DDX18?mDDX18?negatively regulates IFN-? production on mouse-derived cells,and evaluated the effects of mDDX18 knockdown against expression of IFN and IFN-stimulating gene?ISG?induced by virus infection and the proliferation of HSV-1 and VSV in mDDX18+/-mice.The main research contents and results are as follows:1.Interference with mDDX18 promotes virus-induced IFN-? productionSpecific si RNA was used to interfere with the expression of endogenous DDX18 in murine macrophage RAW264.7,and the effect of interference with mDDX18 expression on virus-induced IFN and ISG expression was analyzed.The results showed that interfering with the expression of mDDX18 significantly increased the mRNA level of IFN-? and expression of ISG15 and ISG56 in the downstreamsignaling pathway induced by Sendai virus?Se V?,type 1 human herpes simplex virus?HSV-1?and Vesicular stomatitis virus?VSV?,which confirmed that mDDX18 fuctions in the negative regulation of IFN-? production.2.mDDX18 inhibits IFN-? production independent of its ATPase and helicase activitiesThe full-length c DNA of mDDX18 was cloned fromRAW264.7 cells,and recombinant lentivirus expressing wild-type mDDX18 and its ATPase mutant?mDDX18-K219E?and helicase mutant?mDDX18-S354L?were constructed.The RAW264.7 cells were transducted with recombinant lentivirus,and then stimulated with Se V or VSV.The mRNA level of IFN-? and ISG15 and ISG56 was detected by Real-time PCR,and the content of IFN-? in the cell supernatant was detected by ELISA.The results showed that overexpression of wild-type mDDX18 significantly inhibited the expression of Se V and VSV-induced IFN-?,ISG15 and ISG56,and the inhibitory effects of the two mutants were not significantly different fromthat of wild-type mDDX18,indicating that mDDX18 has the inhibitory effect on IFN-? production and this effect is independent of its helicase activity and ATPase activity.3.Identification and breeding of mDDX18 knockout miceTo further verify the effect of mDDX18 on IFN-? production,a biological company was commissioned to construct mDDX18 knockout F0 generation mice through CRISPR/Cas9 gene editing technology.Through hybridization of F0 generation DDX18+/-mice,we found that the progeny of DDX18+/-mice were all heterozygous?DDX18+/-?or wild-type(DDX18^+/+),and there were no homozygous?DDX18-/-?mice.We speculated that the completely knockout DDX18 results in lethal embryos.Comparing the expression of DDX18 between DDX18+/-mouse and wild-type mouse,we confirmed that the expression of mDDX18 in DDX18+/-mouse were significantly reduced,but there was no significant difference in the body weight and morphology of mice,thus,DDX18+/-mice was used for the subsequent studies in vivo.4.Knockdown of mouse DDX18 promotes virus-induced IFN-? productionDDX18^+/+ and DDX18+/-mouse peritoneal macrophages were separated and infected with Se V,HSV-1 and VSV,respectively.The effect of virus-induced IFN and ISG expression on DDX18^+/+ and DDX18+/-MPMs was analyzed.The results showed that the mRNA level of IFN-?,ISG15 and ISG56 in DDX18+/-MPM cells were significantly up-regulated compared with DDX18^+/+ MPMs under Se V,HSV-1 and VSV infection.The results showed that knockdown of mDDX18 on MPMs up-regulated virus-induced IFN-? production,further confirming that mDDX18 negatively regulates IFN-? production.5.Knockdown of mouse DDX18 promotes anti-viral innate immune response induced by HSV-1Six-week-old wild-type and DDX18+/-mice were infected with HSV-1 via intravenous injection and uninfected controls were set respectively.Blood was collected 18 h after infection,and IFN-? content in serumwas detected by ELISA;mice were sacrificed 3d after infection,brain tissue and lungs were collected.IFN-?,ISG15 and ISG56 mRNA levels in tissues and organs were detected by Real-time PCR,HE staining was used to observe the pathological changes of lungs,and the titer of HSV-1 in brain tissue was detected by TCID50.Eight-week-old wild-type and DDX18+/-mice were infected with HSV-1 via intravenous injection and observed for 12 days on survival.The results showed that under HSV-1 infection,the expression of IFN-?,ISG15 and ISG56 in the brain tissue and lungs of DDX18+/-mice were significantly higher than those in wild-type mice,the content of IFN-? in serumincreased,and HSV-1 titer in brain tissue decreased.Comparing with the uninfected group,HSV-1 infection caused the alveolar wall of DDX18+/-mice and wild-type mice widen significantly,and there was lymphocyte and monocyte infiltration,but HSV-1 infected DDX18+/-mice appeared more monocytes in the lung tissue,suggesting that DDX18+/-mice have a stronger antiviral response against HSV-1 infection.DDX18+/-mice possess higher survival rate than wild-type mice under HSV-1 infection,and the difference of survival between wild-type and DDX18+/-mice was significant.The above results indicate that knocking down DDX18 expression in mice can promote HSV-1 induced IFN-? production and inhibit HSV-1 proliferation,and DDX18+/-mice is more resistant to HSV-1 infection than DDX18^+/+ mice.6.Knockdown of mouse DDX18 promotes anti-viral innate immune response induced by VSVSetting VSV as a case,the effect of knocking down DDX18 on RNA virus-induced natural immunity was evaluated.8-week-old wild-type and DDX18+/-mice were infected with VSV-GFP via intravenous injection,and uninfected controls were set respectively.Blood was collected 18 h after infection,and IFN-? content in serumwas detected by ELISA.The mice were sacrificed 1 d after infection,liver,spleen and lungs were collected,and the mRNA levels of IFN-?,ISG15 and ISG56 in these tissues were detected by Real-time PCR,and the VSV titer in the spleen and liver was detected by TCID50.The results showed that under VSV-GFP infection,the expression of IFN-?,ISG15 and ISG56 in the spleen,liver and lungs of DDX18+/-mice were significantly higher than those in wild-type mice.The content of IFN-? in serumincreased,and VSV titer in spleen and liver decreased.The above results indicate that knocking down the expression of DDX18 in mice can promote VSV-induced IFN-? production and inhibit VSV proliferation.