E3 Ligase RNF5 Inhibits Type Ⅰ Interferon Response In Herpes Simplex Virus Keratitis Through The STING/IRF3 Signaling Pathway

Objective: Herpes simplex keratitis(HSK)caused by herpes simplex virus type I(HSV-1)is the leading cause of infectious blindness in developed countries.HSV infection can occur in any part of the eye and most commonly presents with epithelial or dendritic keratitis.Subsequently,HSV enters the trigeminal ganglion through the corneal nerve and continues to replicate or enter a latent state in the trigeminal ganglion.Under the influence of stress,fever,ultraviolet radiation,and decreased immunity,the virus is reactivated to form recurrent infection.Viral infection activates the transcription factors NF-k B and interferon regulating factor3(IRF3),which cooperate to induce type I interferon and trigger an innate antiviral response.Studies have shown that RING-finger ubiquitin E3 ligase5(RNF5)E3ubiquitin ligase RNF5 interacts with STING in a viral infection-dependent manner.Overexpression of RNF5 suppressed virus-triggered IRF3 activation,IFN-β1expression and cellular antiviral responses,whereas RNF5 inhibition had the opposite effect.Both STING and RNF5 are in the mitochondria and endoplasmic reticulum(ER),and viral infection causes their redistribution,respectively.The E3 ubiquitin ligase RNF5 causes K48-linked ubiquitination of STING at K150 after virus infection,leading to proteasome-mediated degradation of STING and restriction of virus-triggered type I interferon signaling.Sting-deficient mice exhibit impaired production of type I IFN in response to infection with the DNA virus HSV-1 and stimulation with cytosolic DNA.TRIM32 protein regulates type I interferon induction and cellular anti-HSV-1 viral responses by targeting STING proteins to K63-linked ubiquitination.TRIM29 targets STING K48 ubiquitination and degradation to inhibit type I interferon response against HSV-1.In this study,BALB/c mice and human immortalized corneal epithelial cells(HCECs)were used to establish HSV-1 infection models to clarify the abnormal expression of RNF5 during HSV-1 infection.HCEC was used to study the effect of RNF5 silencing and overexpression on type I interferon response during HSV-1 infection in corneal cells.To clarify the effect of silencing RNF5 expression in the cornea on the severity of herpes simplex keratitis,and the effect of silencing RNF5 on the levels of inflammatory cytokines in mice infected with HSV-1.Research method: A stable animal model of herpes simplex keratitis was established by exogenous inoculation of HSV-1 virus into the cornea of BALB/c mice.At the cellular level,a cell model of corneal epithelial cells infected with HSV-1 was established,and the abnormal expression of RNF5 during HSV-1 infection was detected by Western Blot and immunofluorescence.Subsequently,RNF5 was silenced or overexpressed by si RNA or plasmid in corneal epithelial cells to achieve the expected transfection effect,and then HSV-1 was inoculated.Western Blot and q PCR were used to detect the expression changes of STING/IRF3 signaling pathway related proteins during infection.To explore the effect of RNF5 on type I interferon signaling pathway during virus infection.The expression of RNF5 in the mouse cornea was silenced by subconjunctival injection of small interfering RNA(si RNA).After the expected silencing efficiency was achieved,a herpes simplex keratitis model was established,and the severity of keratitis was evaluated by corneal appearance and pathological section staining.Western Blot and q PCR were used to detect the expression of inflammatory cytokines and IFN-β in the cornea of the control group and the silence group.To investigate the effect and mechanism of RNF5 silencing on the severity of keratitis.Results: In the present study,RNF5 expression was found to be significantly increased in corneal tissues and corneal epithelial cells after HSV-1 infection using animal and cellular models.Immunofluorescence staining confirmed that RNF5 was mainly located in the corneal epithelial layer.Silencing and overexpression of RNF5 in corneal epithelial cells and inoculation with HSV-1 showed that the expression of STING,pIRF3,p-TBK1 and the m RNA expression of IFN-β increased after RNF5 silencing.However,the opposite result was obtained after RNF5 overexpression.Subsequently,si RNA was used to silence RNF5 in the mouse cornea to establish a HSK model.The results showed that compared with the si RNA-Control group,the severity of keratitis in the si RNA-RNF5 group was alleviated,and the expression level of IFN-β in the corneal tissue was increased.In addition,the expression of proinflammatory cytokines IL-6 and TNF-α in corneal tissue was significantly reduced,suggesting that RNF5 silencing can effectively promote the expression of type I interferon,inhibit virus replication,alleviate inflammation,and reduce corneal inflammatory injury.The results of the present study suggest that RNF5 limits the type I IFN antiviral response in HSV corneal epithelial dermatitis by inhibiting STING/IRF3 signaling.Conclusion: The expression of RNF5 is increased in the corneal tissues of mice with herpes simplex keratitis.RNF5 was highly expressed in human corneal epithelial cells inoculated with HSV-1.RNF5 inhibits IFN-β secretion induced by HSV-1 infection through STING/IRF3 signaling pathway in human corneal epithelial cells.Silencing RNF5 expression in mouse cornea by si RNA can increase the production of IFN-β and reduce the excessive secretion of proinflammatory cytokines,thereby effectively alleviating the severity of HSK.The present study demonstrates that HSV-1 infection induces elevated RNF5 expression.A lot of RNF5 inhibits STING/IRF3 signaling pathway by ubiquitinating STING and IRF3 degradation,and reduces IFN-β secretion,thereby promoting virus replication and aggravating inflammation.Restriction of RNF5 expression is expected to enhance the host antiviral immune response and alleviate the severity of herpes simplex keratitis.