The Effects And Molecular Mechanism Of Selenoprotein K In Regulating DNA Virus-triggered Innate Immune Responses

BackgroundViral infections pose a serious threat to human health,and host-virus interactions often involve complex regulatory mechanisms.Innate immunity is the first line of defense against viral infection,and is rapidly activated in the early stages of viral infection.Therefore,elucidating the precise regulation between the innate immune system and viruses is particularly important for the prevention and treatment of viral infectious diseases.Stimulator of interferon genes(STING)is an important junction molecule in the innate immune pathway,and is activated by the secondary messenger 2’3’-cyclic GMP-AMP(cGAMP).cGAMP synthase(cGAS)recognizes cytoplasmic DNA and synthesizes cGAMP,which binds to the STING dimer in the endoplasmic reticulum and induces oligomerization of STING,promoting its translocation from the endoplasmic reticulum to the Golgi apparatus,where it activates TANK-binding kinase 1(TBK1)and interferon(IFN)regulatory factor 3(IRF3)and leads to the production of type I IFN.The optimal activation state of STING is crucial for initiating innate immune responses against viral infections and maintaining immune homeostasis.The nutritional status of a host is a key regulator of infectious diseases and regulates complex immune interactions.Selenium is an essential micronutrient in the human body,and its biological functions are primarily mediated by selenoproteins.Most selenoproteins are soluble proteins located in the cytoplasm,and are involved in oxidative stress resistance through regulation of the redox pathway.Eight types of selenoproteins in the selenoprotein family have been identified in the endoplasmic reticulum;however,their functions remain unclear.SELENOK is an endoplasmic reticulum selenoprotein that plays an important role in the immune response.However,the potential role of SELENOK in STING activation and antiviral innate immune response remains unclear.In this study,we used herpes simplex virus type 1(HSV-1)infection as a model to explore the regulatory mechanism of SELENOK in cGAS-STING pathway activation.ObjectiveThe objective of this study was to analyze the potential role of SELENOK in antiviral innate immunity and its regulatory mechanism in cGAS-STING pathway activation.The findings provide new insights into the STING activation mechanism and a theoretical basis for discovering new targets for antiviral immunotherapy.Methods and results1.SELENOK is the only endoplasmic reticulum selenoprotein that specifically promotes cGAS-STING pathway activationUsing the small interfering RNA corresponding to each endoplasmic reticulum selenoprotein,a mouse gene knockout model of HSV-1-infected primary peritoneal macrophages was constructed,and unbiased screening was conducted using real-time polymerase chain reaction(PCR).Selenok knockdown significantly reduced the expression of HSV-1 infection-induced Ifnb mRNA.Other endoplasmic reticulum selenoproteins did not show such effects.This suggests that Selenok may be involved in regulating the antiviral innate immune response induced by HSV-1 infection.2.SELENOK promotes the activation of the cGAS-STING pathwayEnzyme-linked immunosorbent assay(ELISA),real-time PCR,and western blot analyses showed that Selenok deficiency or knockdown inhibited HSV-1,and ISD induced the secretion of IFN-β and interleukin(IL)-6,expression of Ifnb mRNA,phosphorylation of TBK1,IRF3,and Signal transducer and activator of transcription 1(STAT1),and downstream expression of IFN-stimulated genes(ISGs).However,SeV and CpG oligodeoxynucleotides(CpG ODNs)were not significantly affected.These results indicate that Selenok deficiency or knockdown selectively inhibits the cGAS-STING pathway,but has no significant effect on the retinoic acid-inducible gene Ⅰ(RIG-I)and Toll-like receptor 9(TLR9)pathways.3.Selenok deficiency inhibits innate immune response induced by HSV-1 in vivoTo investigate the physiological and pathological correlations between SELENOK and HSV-1 infection in vivo,a model of intraperitoneal and intracerebral HSV-1 infection was established using wild-type and Selenok-deficient mice.Compared with the control group,Selenok-deficient mice showed a significant decrease in type Ⅰ interferon response,increased virus replication,and damage to lung and brain tissues.Additionally,survival rate experiments revealed that resistance to viral infections was significantly decreased in Selenok-deficient mice.These results indicate that SELENOK plays a crucial regulatory role in resistance to HSV-1 infection.4.SELENOK promotes STING activationELISA showed that the regulation of SELENOK in the cGAS-STING pathway was not caused by the impairment of receptor-mediated calcium flux due to Selenok deficiency.Luciferase reporter gene assay data showed that cGAS and STING could significantly promote IFN-β expression,whereas TBK1 could not,indicating that Selenok may target cGAS or STING.Western blot and ELISA analyses showed that Selenok deficiency reduced STING phosphorylation levels but did not affect the binding of cGAS to ISD or cGAMP generation.ELISA,real-time PCR,and western blot analyses showed that Selenok deficiency or knockdown inhibited the secretion of IFN-β and IL-6,Ifnb mRNA expression,and the expression of downstream phosphorylation molecules and ISGs induced by STING activators cGAMP and 5,6-dimethylxanthenone-4-acetic acid(DMXAA).The results of the STING-activated in vivo experimental model showed that,compared to the control group,serum IFN-β and IL-6 secretion in Selenok-deficient mice significantly decreased.These results indicate that SELENOK targets STING instead of cGAS.5.SELENOK interacts with STINGThe results of endogenous and exogenous immunocoprecipitation experiments showed that SELENOK interacted with STING.Immunofluorescence experiments showed that SELENOK and STING colocalized.After STING activation,the SELENOK-STING complex was translocated to the Golgi apparatus.An immunocoprecipitation experiment performed using truncated SELENOK and STING showed that SELENOK and STING interacted through the endoplasmic reticulum domain.6.SELENOK enhances the activation of the cGAS-STING pathway by promoting STING oligomerizationWestern blot analysis showed that Selenok deficiency did not affect STING dimerization.Irrespective of the presence or absence of BFA pretreatment,Selenok deficiency significantly inhibited STING oligomerization,indicating that SELENOK promotes STING oligomerization before entering the Golgi apparatus.Immunofluorescence experiments showed a significant decrease in STING translocation to coat protein complex II vesicles or Golgi apparatus in Selenok-deficient MEFs,indicating that Selenok deficiency inhibited STING translocation.Western blot showed that high SELENOK expression in HEK 293T cells could also promote STING oligomerization without cGAMP stimulation and could not be inhibited by BFA.Selenok deficiency did not inhibit STING palmitoylation,and mutation of the STING palmitoylation site did not affect SELENOK-induced STING oligomerization.These results indicate that SELENOK promotes STING oligomerization,thus enhancing the activation of the cGAS-STING pathway.7.IFN-β promotes the expression of SELENOKELISA and real-time PCR analyses showed that selenium promoted a type I IFN response after HSV-1 infection and inhibited viral replication.Real-time PCR and western blot analysis showed that selenium promotes the expression of SELENOK,while HSV-1 and IFN-β could also increase SELENOK expression at the cellular and mouse tissue levels.The increase in SELENOK expression was not regulated at the transcriptional level,but IFN-βpromoted the protein synthesis of SELENOK,indicating that the secretion of HSV-1 infection-mediated IFN-β induces increased SELENOK expression,thus providing a positive feedback effect for STING-dependent host immune defense.ConclusionThis study confirmed that SELENOK promotes an innate immune response against HSV-1 and that its expression increases during viral infection,thus forming a positive feedback loop.SELENOK and STING promote the oligomerization of STING through interaction with the endoplasmic reticulum domain,allowing STING to translocate from the endoplasmic reticulum to the Golgi apparatus and activate the cGAS-STING pathway.