This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Legionella pneumophila causes a severe pneumonia known as Legionnaires’ disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1’), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host–pathogen interactions.

Main protease and papain-like protease (PLpro) are essential coronaviral enzymes required for polypeptide processing during viral maturation. PLpro additionally cleaves proteinous post-translational modifications from host proteins to evade anti-viral immune responses. Here, we provide biochemical, structural and functional characterizations of PLpro from SARS-CoV-2 (PLproCoV2) and reveal differences to that of SARS (PLproSARS) in controlling interferon (IFN) and NF-kB pathways. PLproCoV2 and PLproSARS share 83% sequence identity, yet they differ in their host substrate preferences: PLproCoV2 predominantly cleaves the ubiquitin-like protein ISG15 off from host proteins, while PLproSARS preferentially targets ubiquitin chains. The crystal structure of PLproCoV2 in complex with ISG15 explains the affinity and higher specificity through distinctive binding to ISG15’s unique amino-terminal ubiquitin-like domain, and enabled the identification of GRL-0617 as a non-covalent candidate inhibitor for PLproCoV2. In human cells, PLproCoV2 cleaves ISG15 from interferon responsive factor 3 (IRF3), blocks its nuclear translocation, and reduces type I interferon responses, whereas PLproSARS preferentially mediates deubiquitination of critical components of the NF-kB pathway. Pharmacological inhibition of PLproCoV2 blocks the virus-induced cytopathogenic effect upon infection with SARS-CoV-2, fosters the anti- viral interferon pathway and reduces viral release from infected cells. We propose that therapeutic targeting of PLproCoV2 can suppress SARS-CoV-2 infection and promote anti-viral immunity.

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, non-hospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling anti-viral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.

Legionella pneumophila is a gram-negative pathogenic bacterium that causes 19Legionaries' disease. The Legionella genome codes more than 300 effector proteins able to 20 modulate host-pathogen interactions during infection. Among them are also enzymes altering the 21 host-ubiquitination system including bacterial ligases and deubiquitinases. In this study, based on 22 homology-detection screening on 305 Legionella effector proteins, we identified two Legionella 23 OTU-like deubiquitinases (LOT; LotB (Lpg1621/Ceg23) and LotC (Lpg2529), LotA 24 (Lpg2248/Lem21) is already known). A crystal structure of LotC catalytic core (LotC14-310) was 25 determined at 2.4 Å and compared with other OTU deubiquitinases, including LotB. Unlike the 26 classical OTU-family, the structures of Legionella OTU-family (LotB and LotC) shows an 27 extended helical lobe between the Cys-loop and the variable loop, which define a novel class of 28 OTU-deubiquitinase. Despite structural differences in their helical lobes, both LotB and LotC 29 interact with ubiquitin. LotB has an additional ubiquitin binding site (S1') enabling specific 30 cleavage of Lys63-linked poly-ubiquitin chains. By contrast, LotC only contains the S1 site and 31 cleaves different species of ubiquitin chains. MS analysis of catalytically inactive LotB and LotC 32 identified different categories of host-substrates for these two related DUBs. Together, our results 33 provide new structural insights of bacterial OTU deubiquitinases and indicate distinct roles of 34 bacterial deubiquitinases in host-pathogen interactions. 35