The Otubain Deubiquitinase YOD1 Fine-tunes MAVS Mediated Innate Immunity

Objectives:Innate immunity is the first line of host defense against viral infections.In recent years,related studies have confirmed that ubiquitination and deubiquitination modifications play an important role in innate immunity.YOD1 is a member of the Otubain family,however the function of YOD1 in innate immunity is not clear.Considering that YOD1 is a deubiquitinating enzyme,investigate whether YOD1 involved the antiviral innate immunity response through its deubiquitinating function,and to explore the function of YOD1 in MAVS signaling and uncover the molecular mechanism,provide new insights into cellular antiviral response.Methods:1.To identify the potential regulators of MAVS signaling,we overexpressed Flag-tagged MAVS in HEK293 T cells and performed mass spectrometric analysis.2.We employed q RT-PCR assays and Western Blot to check if the expression of YOD1 was correlated with infection by RNA virus in several cell lines.3.We constructed the YOD1-WT and YOD1-C160 S plasmids and designed two human YOD1 si RNAs,transfected into HEK293 T cells upon infection of VSV-GFP virus,to determine the biological function of YOD1 in regulating virus amplification.4.To explore whether YOD1 played a role in regulating MAVS-mediated IFN-?signaling,we assess the expression level of IFNs and ISGs through q RT-PCR assays and reporter assays.5.To further explore the molecular regulatory mechanism of YOD1,we checked the interaction between YOD1 and MAVS in mammalian cells by transient transfection and coimmunoprecipitation experiments.To determine which domain(s)of YOD1 and MAVS were required for their interaction,we created several truncate variants and evaluated them in coimmunoprecipitation experiments.6.To better understand the interaction between MAVS and YOD1,we carried out confocal microscopy assay and employed cell fractionation and immunoblot analysis to monitor YOD1 localization with or without Sendai virus infection.7.We next focused on investigating whether YOD1 could influence on the status of MAVS ubiquitin by means of in vivo ubiquitination assays and to distinguish which type of lysine linkage ubiquitin of MAVS was regulated by YOD1,we also employed ubiquitin mutants with only one lysine at position 48 or 63 available for conjugation(K48 or K63).8.We performed the SDD-AGE assay to determine whether YOD1 influenced MAVS aggregates.Results:MAVS is a critical adaptor required for activating an innate antiviral immune response against viral infection.The activation of MAVS requires modification of the Lys63-linked ubiquitination and formation of prion-like aggregates.However,the molecular mechanisms regulating MAVS activity remain largely unveiled.Here we identified a deubiquitinase YOD1,also known as a member of the ovarian tumor(OTU)family,as a negative regulator of MAVS activation in both human and murine cells.YOD1 was recruited to mitochondria to interact with MAVS through its UBX and Znf domains after viral infection.Subsequently,YOD1 cleaved the K63-linked ubiquitination and abrogated the formation of prion-like aggregates of MAVS,which led to attenuation of IRF3 and P65 activation and IFN? production.Knockdown of YOD1 potentiated IRF3 and P65 activation,IFN? production and antiviral innate immune response to RNA virus.Our findings thus provided novel insights into the regulatory cascade of the cellular antiviral response through YOD1-mediatd K63-linked deubiquitination and aggregation of MAVS.Conclusions1.RNA virus infection induces YOD1 expression.2.YOD1 negatively regulated MAVS-mediated IFN? signaling.3.YOD1 interacted with MAVS.4.YOD1 cleaved the K63-linked ubiquitination chains on MAVS.5.YOD1 suppressed the aggregation of MAVS.6.YOD1 is involved in cellular antiviral response.