The Mechanism Of The Promotion Of The Innate Immune Response To Viral Infection By REC8

Innate immunity is the host’s first line of defense against viral invasion.When the virus infects host cells,its viral nucleic acid can be recognized by the pattern recognition receptors of the host cell,and then activate the host innate immune response,inducing the expression of interferons,pro-inflammatory cytokines,chemokines,and interferon-stimulating factors,thereby exerting an antiviral effect.Interferon-stimulated genes are the main factors for the host to resist virus invasion.Only a small number of ISGs have been clearly explored.The antiviral and specific functional mechanisms of most ISGs are still unclear.REC8(REC8 meiotic recombination protein),one of ISGs,was found in the RNA sequencing.REC8 may play an important role in viral infection.The following studies has been carried out for this purpose:First,interferon or virus induces the expression of REC8.We treated HLCZ01 cells with IFN-α,interferon-stimulated RNA-Poly I:C or viruses such as VSV,NDV or HSV.The data showed that interferon or virus could induce the expression of REC8.To determine whether the induction of REC8 is dependent on the activation of JAK/STAT signaling pathway by IFN,we knocked down the int erferon receptor-IFNAR1 in HLCZ01 cells,and then treated the cells with IFN-α,Poly I:C,VSV,NDV,or HSV.The data showed that silencing IFNR1 inhibited the induction of REC8 by IFN-α or viral infection,suggesting that the induction of REC8 by viral infe ction depends on the activation of JAK/STAT signaling pathway by IFN.All the data indicates that REC8 may play an important role in viral infection.Second,REC8 promotes the innate immune response to viral infection and subsequently inhibits viral replication.To test the antiviral activity of REC8,we silenced or overexpressed REC8 in HLCZ01 cells.The data showed that overexpression of REC8 inhibited the replication of VSV,NDV,HCV or HSV,while silencing of REC8 enhanced viral replication.To examine whether REC8 inhibits viral replication by promoting the innate immune response,we treated HLCZ01 cells with RNA nucleic acid mimics Poly:C or HCV 3’UTR RNA.The data showed that overexpression of REC8 augmented the expression of interferon(IFN-β,IL28 A,IL29)and interferon-stimulated genes(ISG12a,ISG15),while silencing of REC8 attenuated the expression of IFNs and ISGs.Moreover,overexpression or silencing of REC8 promoted or inhibited the expression of IFNs and ISGs induced by RNA virus(VSV or NDV)or DNA virus(HSV)infection,supporting that REC8 promotes viral-trigged innate immune response.Third,REC8 promotes the activation of IRF3 and NF-κB signaling by interacting with MAVS and STING.IRF3 and NF-κB signaling are the main signaling pathways for the host to activate the innate immune response to produce antiviral factors.To explore whether REC8 regulates IRF3 and NF-κB signaling,we overexpressed or silenced REC8 in HLCZ01,A549,and THP-1 cells.The data showed that REC8 augmented the phosphorylation of TBK1,IRF3,P65,STAT1 and other proteins triggered by virus such as VSV,NDV or HSV,while silencing REC8 impaired the virus-induced the phosphorylation of IRF3,P65,STAT1,etc.These results demonstrated that REC8 promotes the activation of IRF3 and NF-κB signaling pathway by viral infection.Luciferase reporter gene assay,co-immunoprecipitation and immunofluorescence staining results showed that REC8 targeted and interacted with MAVS or STING,the key cytoplasmic adaptor protein of innate immunity.Fourth,viral infection induces the SUMOylation of REC8 and promotes the translocation of REC8 from the nucleus to the cytoplasm.REC8 is mainly located in the nucleus in the resting state.To explore whether the localization of REC8 changes du ring virus infection,we carried out nucleocytoplasmic separation assays and immunofluorescence assay.The results showed that upon viral infection,REC8 increased in the cytoplasm while it decreased in the nucleus.These data supported that REC8 undergoes nucleo-cytoplasmic transfer under virus infection.SUMOylation can affect protein transport.Viral infection triggered the SUMOylation of REC8 at K30 and K530.Silencing UBC9 impaired the SUMOylation of REC8 and subsequently attenuated the nuclear export of REC8,thereby inhibiting the innate immune response to viral infection.Fifth,REC8 inhibits the ubiquitination and degradation of MAVS and STING.The activity and stability of MAVS and STING protein directly affect the activation of downstream signaling.REC8 inhibited the degradation of MAVS and STING through proteasome system.The ubiquitination results showed that REC8 inhibited the K48-dependent ubiquitination of MAVS at K362 and STING at K150 and K370,and subsequently impaired the degradation of MAVS and STING.Co-IP and ubiquitination results showed that REC8 interacted with RNF5,the common E3 ligase of MAVS and STING.Moreover,REC8 inhibited the ubiquitination and degradation of MAVS and STING mediated by RNF5.In conclusion,REC8 is found to be an new ISG in this study.Under virus infection,REC8 can be SUMOylated,and translocated from the nucleus to the cytoplasm.Cytoplasmic REC8 interacts with MAVS or STING to inhibit the K48-dependent ubiquitination and degradation of MAVS and STING triggered by RNF5,and stabilize MAVS and STING,thereby promoting the innate immune response to viral replication.This study demonstrates role of REC8 in viral infection and elucidates the mechanism by which REC8 promotes the innate immune response to viral.The study discloses a novel mechanism by which the host stabilizes MAVS and STING protein under viral infection.