The Mechanisms Of RNF26Mediated Regulation Of Cellular Antiviral Signal Transduction And The Screening Of Peroxisome-associated Antiviral Proteins

Innate immune system is the frontier of host defense against the invasion of pathogens. The recognition of pathogens depends on host pattern-recognition receptors (PRRs). Nucleic acid from invading viruses can be detected by PRRs and triggers a series of signaling events that lead to the induction of type I interferons, which plays a central role in autoimmune diseases as well as protective immune responses against viruses, respectively. There has been huge progress in characterizing viral nucleic acid-triggered signaling pathways that result in transcriptional activation of type IIFN genes. Retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene5(MDA5) detect viral RNA and recruit the adaptors including virus-induced signaling adaptor (VISA, also called MAVS, IPS-1and Cardif) and mediator of IRF3activation (MITA, also known as STING, MPYS and ERIS) to activate the transcription factors nuclear factor kappa B (NF-κB) and interferon regulatory factor (IRF)3or IRF7, leading to transcriptional induction of the genes encoding type I IFNs and other antiviral effectors. Viral DNA can be detected by a number of cytosolic DNA sensors. These sensors exclusively depend on the adaptor MITA to activate NF-κB and IRF3/7and lead to subsequent induction of type I IFNs.MITA is critically required for virus-triggered induction of type I interferons (IFNs) and innate antiviral response. The throrough understading of how post-translational modifications regulate the activity of MITA stays challenging. In the present study, we show that the E3ubiquitin ligase RING finger protein26(RNF26) promotes K11-linked polyubiquitination of MITA at lysine150. On one hand, RNF26protects MITA from RNF5-mediated K48-linked polyubiquitination and degradation that is required for quick and efficient type I IFN responses. On the other, RNF26is required to limit excessive type I IFN responses by promoting autophagic degradation of IRF3. Consistently, knockdown of RNF26inhibited the expression of IFNB1gene in various cells at the early phase and promoted it at the late phase of viral infection, respectively. Our findings thus suggest that RNF26temporally regulates innate antiviral response by two distinct mechanisms. This work provide new insights into the molecular regulation of cellular antiviral responses.Another critical adaptor in virus-induced type Ⅰ IFNs signaling, VISA, was first reported to be localized on mitochondria. Recent studies suggested that VISA is also located on peroxisomes. Besides, peroxisomal and mitochondrial VISA mediate different antiviral effects during different phase of viral infection. The finding of peroxisomes, as an important site of antiviral signal transduction, leads to the screening of peroxisome-associated proteins which involve in innate antiviral responses. In the present study, we enrich the peroxisomes from SeV-stimulated or untreated HepG2cells and use stable-isotope dimethylation labeling method for relative quantification. Reproducible results of mass spectrometry analysis show that67of695credible proteins on peroxisomes and associated subcellular fractions were upregulated after SeV infection, while73were downregulated. Among them, some genes were reported to play a role in antiviral responses. The function of some candidates, in innate immunity, is verified in subsequent experiments. We effectively enrich the peroxisomes of HepG2cells and screen some proteins involved in antiviral innate immune responses. This work would lead us to a better understanding of the mechanism of antiviral innate immunity.