Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression

Author summary Outbreaks of the picornavirus foot-and-mouth disease virus (FMDV) have significant consequences for animal health and product safety and place a major economic burden on the global livestock industry. Understanding how this notorious animal pathogen suppresses the antiviral type I interferon (IFN-alpha/beta) response may help to develop countermeasures to control FMDV infections. FMDV suppresses the IFN-alpha/beta response through the activity of its Leader protein (L-pro), a protease that can cleave host cell proteins. L(pro)was also shown to have deubiquitinase and deISGylase activity, raising the possibility that L(pro)suppresses IFN-alpha/beta by removing ubiquitin and/or ISG15, two posttranslational modifications that can regulate the activation, interactions and localization of (signaling) proteins. Here, we show that TBK1 and MAVS, two signaling proteins that are important for activation of IFN-alpha/beta gene transcription, are cleaved by L-pro. By generating L(pro)mutants lacking either of these two activities, we demonstrate that L-pro's ability to cleave signaling proteins, but not its deubiquitination/deISGylase activity, correlates with suppression of IFN-beta gene transcription. The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-alpha/beta) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (L-pro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-alpha/beta gene transcription; however, the exact mechanism is unknown. The proteolytic activity of L(pro)is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-kappa B. In addition, L(pro)has been demonstrated to have deubiquitination/deISGylation activity. L-pro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-alpha/beta gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by L(pro)in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing L-pro.In vitrocleavage experiments revealed that L(pro)cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-L-pro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK alpha V beta 6 cells. We set out to dissect L-pro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity, to determine their relative contributions to the reduction of IFN-alpha/beta gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of L(pro)in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of L-pro. Characterization of the effects of these mutations revealed that L-pro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-beta gene transcription

The tumor suppressor TMEM127 is a Nedd4-family E3 ligase adaptor required by Salmonella SteD to ubiquitinate and degrade MHC class II molecules

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII β chain. Here, through a genome-wide mutant screen of human antigen-presenting cells, we show that the NEDD4 family HECT E3 ubiquitin ligase WWP2 and a tumor-suppressing transmembrane protein of unknown biochemical function, TMEM127, are required for SteD-dependent ubiquitination of mMHCII. Although evidently not involved in normal regulation of mMHCII, TMEM127 was essential for SteD to suppress both mMHCII antigen presentation in mouse dendritic cells and MHCII-dependent CD4+ T cell activation. We found that TMEM127 contains a canonical PPxY motif, which was required for binding to WWP2. SteD bound to TMEM127 and enabled TMEM127 to interact with and induce ubiquitination of mature MHCII. Furthermore, SteD also underwent TMEM127- and WWP2-dependent ubiquitination, which both contributed to its degradation and augmented its activity on mMHCII

Coccidioidomycosis among Workers at an Archeological Site, Northeastern Utah

In 2001, an outbreak of acute respiratory disease occurred among persons working at a Native American archeological site at Dinosaur National Monument in northeastern Utah. Epidemiologic and environmental investigations were undertaken to determine the cause of the outbreak. A clinical case was defined by the presence of at least two of the following symptoms: self-reported fever, shortness of breath, or cough. Ten workers met the clinical case definition; 9 had serologic confirmation of coccidioidomycosis, and 8 were hospitalized. All 10 were present during sifting of dirt through screens on June 19; symptoms began 9–12 days later (median 10). Coccidioidomycosis also developed in a worker at the site in September 2001. A serosurvey among 40 other Dinosaur National Monument workers did not find serologic evidence of recent infection. This outbreak documents a new endemic focus of coccidioidomycosis, extending northward its known geographic distribution in Utah by approximately 200 miles

Insights into ubiquitin chain architecture using Ub-clipping

Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation(1-3). Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lb(pro)*, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lb(pro)* retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10-20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code

Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies

Chemical Immunolog