The Mechanism Of USP15 Regulation In Type â…  Interferon And Global Analysis Of Ubiquitome In PRRSV-Infected Cells

Ubiquitination is a key post-translational modification that involves the covalent attachment of one or more ubiquitin monomers to lysine residues of target proteins, and is a three-step enzymatic cascade mediated by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. Emerging evidence indicates that ubiquitination plays a pivotal role in a myriad of biological processes, including cell-cycle regulation, cell apoptosis, DNA repair, protin-protein interaction and cell signal transduction.Type I interferon is the focus of innate immunity for being as an important antiviral factor against pathogens. Aberrant production of type I interferon may result in the development of immunopathological conditions. Thus, tight regulation of type I interferon signaling is of great significance for maintaining the immune homeostasis. Ubiquitination is an important post-translational modification of proteins and plays a key role in the production of interferon and signaling transduction. Ubiquitin-specific proteases(USPs) represent the bulk of deubiquitylating enzymes(DUBs) and receive extensive attentions for the diversity in structure and function. In this study, we dissect the specific mechanism by which USP15 regulates type I interferon signaling pathway, laying the theoretical foundation for the study of biological functions of USPs.In addition to regulating mutiple physiological functions, ubiquitination is also involved in the regulation of viral replication and proliferation. For instance, bovine viral diarrhea virus genome has ubiquitin and ubiquitin-like genes; Rotavirus utilizes the ubiquitin-proteasome system to modulate its replication; Human immunodeficiency virus-encoded proteins undergo ubiquitination modification, and some viral genes encode E3 ubiquitin-protein ligases or regulate cellular protein ubiquitination by other means. Porcine reproductive and respiratory syndrome virus(PRRSV) has devastated the swine industry worldwide since the late 1980 s and has complicated pathogenicity. The role ubiquitination plays in PRRSV infection is not clearly described. In this study, anti-ubiquitination-based enrichment and liquid chromatography-tandem mass spectrometry were applied to comprehensively investigate the global ubiquitination events triggered by PRRSV infection in pulmonary alveolar macrophages(PAMs), providing a foundation for the analysis of the significant and unique roles of ubiquitination in PRRSV infection and pathogenicity. The main research works were as following: 1. The mechanism of USP15 regulation in type I interferonAfter screening USPs for regulation in interferon(IFN), we found the activity of ISRE promoter is up-regulated in USP15-knocked-down cells, then we further validated whether USP15 can inhibit the production of IFN. We found Sendai virus(SEV)-induced IFN-β expression(activity of IFN-β report plasmid, transcription of endogenous IFNB1 gene, production of IFN-β proteins), IRF3 and NF-κB promter activity as well as their phosphorylation, ISRE promoter activity and the production of ISGs were higher in the USP15-knocked-down cells than in the control-siRNA-treated cells. On the contrary, overexpression of USP15 significantly suppressed the virus-induced type I IFN signaling pathway. We further investigated the effect of USP15 on antiviral responses by monitoring the VSV-GFP expression under fluorescence microscope. Remarkably, overexpression of USP15 reduced IFN-β expression. Furthermore, we confirmed USP15 is a potent DUB that recognizes and degrades polyubiquitin chains in vitro and in vivo, and its catalytic activity is dependent on Cys269 and His862. Consistent with the negative regulatory role of USP15 in type I IFN signaling pathway, USP15 substantially reduced RIG-I polyubiquitination, and specifically towards the Lys63-linked polyubiquitin. The catalytic mutants, which have lost their deubiquitinating activity, reduced but did not abolish the capability of USP15 to block the activation of IFN-b, indicating the DUB activity of USP15 is not strictly required for inhibition of antiviral IFN expression. We also found USP15 interacts with RIG and then impairs the recruitment of IPS-1 by RIG-I. Additionally, the catalytic mutants of USP15 also disrupted RIG-I-IPS-1interaction through binding to RIG-I, highlighting the notion of catalytic-activity-independent mechanism of IFN antagonism. Two mechanisms by which USP15 plays a role in down-regulating the antiviral innate immune response are: 1. USP15 removes Lys-63 linked polyubiquitination conjugates from RIG-I. 2. USP15 sequesters the interaction between RIG-I and IPS-1 by acting as a competitor of IPS-1 for RIG-I binding. 2. Global analysis of ubiquitome in PRRSV-infected cellsPAMs were inoculated with PRRSV strain WUH3, and infected cells were collected for further treatments 36 h postinfection. Label-free approach coupled with quantitative LC-MS/MS were used to identify host proteins that exhibit ubiquitination level alteration during PRRSV infection and replication. In total, a number of significantly differentially expressed Kub sites on proteins were obtained(431 Kub sites on 315 proteins were quantified as up-regulated and 552 Kub sites on 402 proteins were down-regulated). By integrating highly sensitive MS and bioinformatics tools, we profiled subcellular locations, biological processes and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways of the proteins exhibiting ubiquitination level alteration during PRRSV infection. The bioinformatic analysis showed that these differentially regulated ubiquitinated proteins were involved in proteasome, protein localization and catabolic process. The position-specific frequencies of the amino acid residues surrounding ubiquitinated lysine residues of target proteins were characterized. Totally three motifs respectively named KubXXP, RXXXXLXKub and KubQ were identified. Crosstalk analysis between global differentially expressed proteome and ubiquitome indicated that the proteome and ubiquitome is not obvious related uopn PRRSV infection. More importantly, many ubiquitination level altered proteins were associated with interferon and NF-κB signaling pathway, including TRAF6, IL-1β, HMGB1, JAK1, Mx2 and ISG15. The results highlight that alteration in ubiquitination level plays an important role in PRRSV-regulated interferon and inflammatory responses. Meanwhile, we analyzed PRRSV proteins for ubiquitylated residues and 64 ubiquitylated residues on 15 viral proteins(nsp1β、nsp2、nsp3、nsp4、nsp7、nsp8、nsp9、nsp10、nsp11、nsp12、ORF2a、ORF3、ORF5、ORF6、ORF7)were identified, including proteins involved in RNA synthesis, neutralizing antibody elicitation and virus infectivity. Further studies diaplayed that treatment of cells with proteasome inhibitors significantly reduced the viral titer. These data indicated ubiquitination process may plays a key role in PRRSV transcription, replication and protein synthesis.