Fragment-Based Discovery of Novel MUS81 Inhibitors.

MUS81 is a structure-selective endonuclease that cleaves various branched DNA structures arising from natural physiological processes such as homologous recombination and mitosis. Due to this, MUS81 is able to relieve replication stress, and its function has been reported to be critical to the survival of many cancers, particularly those with dysfunctional DNA-repair machinery. There is therefore interest in MUS81 as a cancer drug target, yet there are currently few small molecule inhibitors of this enzyme reported, and no liganded crystal structures are available to guide hit optimization. Here we report the fragment-based discovery of novel small molecule MUS81 inhibitors with sub-µM biochemical activity. These inhibitors were used to develop a novel crystal system, providing the first structural insight into the inhibition of MUS81 with small molecules.

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates.

TRIM7 Restricts Coxsackievirus and Norovirus Infection by Detecting the C-Terminal Glutamine Generated by 3C Protease Processing.

TRIM7 catalyzes the ubiquitination of multiple substrates with unrelated biological functions. This cross-reactivity is at odds with the specificity usually displayed by enzymes, including ubiquitin ligases. Here we show that TRIM7's extreme substrate promiscuity is due to a highly unusual binding mechanism, in which the PRYSPRY domain captures any ligand with a C-terminal helix that terminates in a hydrophobic residue followed by a glutamine. Many of the non-structural proteins found in RNA viruses contain C-terminal glutamines as a result of polyprotein cleavage by 3C protease. This viral processing strategy generates novel substrates for TRIM7 and explains its ability to inhibit Coxsackie virus and norovirus replication. In addition to viral proteins, cellular proteins such as glycogenin have evolved C-termini that make them a TRIM7 substrate. The 'helix-ΦQ' degron motif recognized by TRIM7 is reminiscent of the N-end degron system and is found in ~1% of cellular proteins. These features, together with TRIM7's restricted tissue expression and lack of immune regulation, suggest that viral restriction may not be its physiological function.

Critical Care Workers Have Lower Seroprevalence of SARS-CoV-2 IgG Compared with Non-patient Facing Staff in First Wave of COVID19.

INTRODUCTION: In early 2020, at first surge of the coronavirus disease 2019 (COVID-19) pandemic, many health care workers (HCW) were re-deployed to critical care environments to support intensive care teams looking after patients with severe COVID-19. There was considerable anxiety of increased risk of COVID-19 for these staff. To determine whether critical care HCW were at increased risk of hospital acquired infection, we explored the relationship between workplace, patient facing role and evidence of immune exposure to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a quaternary hospital providing a regional critical care response. Routine viral surveillance was not available at this time. METHODS: We screened over 500 HCW (25% of the total workforce) for history of clinical symptoms of possible COVID19, assigning a symptom severity score, and quantified SARS-CoV-2 serum antibodies as evidence of immune exposure to the virus. RESULTS: Whilst 45% of the cohort reported symptoms that they consider may have represented COVID-19, 14% had evidence of immune exposure. Staffs in patient facing critical care roles were least likely to be seropositive (9%) and staff working in non-patient facing roles most likely to be seropositive (22%). Anosmia and fever were the most discriminating symptoms for seropositive status. Older males presented with more severe symptoms. Of the 12 staff screened positive by nasal swab (10 symptomatic), 3 showed no evidence of seroconversion in convalescence. CONCLUSIONS: Patient facing staff working in critical care do not appear to be at increased risk of hospital acquired infection however the risk of nosocomial infection from non-patient facing staff may be more significant than previous recognised. Most symptoms ascribed to possible COVID-19 were found to have no evidence of immune exposure however seroprevalence may underrepresent infection frequency. Older male staff were at the greatest risk of more severe symptoms.

Target-induced clustering activates Trim-Away of pathogens and proteins.

Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyglutamine tracts and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.

Comparative assessment of multiple COVID-19 serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings.

There is a clear requirement for an accurate SARS-CoV-2 antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. We therefore evaluated the performance of a variety of antibody testing technologies and their potential use as diagnostic tools. Highly specific in-house ELISAs were developed for the detection of anti-spike (S), -receptor binding domain (RBD) and -nucleocapsid (N) antibodies and used for the cross-comparison of ten commercial serological assays-a chemiluminescence-based platform, two ELISAs and seven colloidal gold lateral flow immunoassays (LFIAs)-on an identical panel of 110 SARS-CoV-2-positive samples and 50 pre-pandemic negatives. There was a wide variation in the performance of the different platforms, with specificity ranging from 82% to 100%, and overall sensitivity from 60.9% to 87.3%. However, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over 95% seen in several tests when assessing samples from more than 20 days post onset of symptoms. Furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. Rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of SARS-CoV-2 infections.

Antibody fragments structurally enable a drug-discovery campaign on the cancer target Mcl-1.

Apoptosis is a crucial process by which multicellular organisms control tissue growth, removal and inflammation. Disruption of the normal apoptotic function is often observed in cancer, where cell death is avoided by the overexpression of anti-apoptotic proteins of the Bcl-2 (B-cell lymphoma 2) family, including Mcl-1 (myeloid cell leukaemia 1). This makes Mcl-1 a potential target for drug therapy, through which normal apoptosis may be restored by inhibiting the protective function of Mcl-1. Here, the discovery and biophysical properties of an anti-Mcl-1 antibody fragment are described and the utility of both the scFv and Fab are demonstrated in generating an Mcl-1 crystal system amenable to iterative structure-guided drug design.

NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula.

To overcome nitrogen deficiencies in the soil, legumes enter symbioses with rhizobial bacteria that convert atmospheric nitrogen into ammonium. Rhizobia are accommodated as endosymbionts within lateral root organs called nodules that initiate from the inner layers of Medicago truncatula roots in response to rhizobial perception. In contrast, lateral roots emerge from predefined founder cells as an adaptive response to environmental stimuli, including water and nutrient availability. CYTOKININ RESPONSE 1 (CRE1)-mediated signaling in the pericycle and in the cortex is necessary and sufficient for nodulation, whereas cytokinin is antagonistic to lateral root development, with cre1 showing increased lateral root emergence and decreased nodulation. To better understand the relatedness between nodule and lateral root development, we undertook a comparative analysis of these two root developmental programs. Here, we demonstrate that despite differential induction, lateral roots and nodules share overlapping developmental programs, with mutants in LOB-DOMAIN PROTEIN 16 (LBD16) showing equivalent defects in nodule and lateral root initiation. The cytokinin-inducible transcription factor NODULE INCEPTION (NIN) allows induction of this program during nodulation through activation of LBD16 that promotes auxin biosynthesis via transcriptional induction of STYLISH (STY) and YUCCAs (YUC). We conclude that cytokinin facilitates local auxin accumulation through NIN promotion of LBD16, which activates a nodule developmental program overlapping with that induced during lateral root initiation.

Murine gammaherpesvirus targets type I IFN receptor but not type III IFN receptor early in infection.

The innate immune response represents a primary line of defense against invading viral pathogens. Since epithelial cells are the primary site of gammaherpesvirus replication during infection in vivo and there are no information on activity of IFN-III signaling against gammaherpesviruses in this cell type, in present study, we evaluated the expression profile and virus-host interactions in mouse mammary epithelial cell (NMuMG) infected with three strains of murine gammaherpesvirus, MHV-68, MHV-72 and MHV-4556. Studying three strains of murine gammaherpesvirus, which differ in nucleotide sequence of some structural and non-structural genes, allowed us to compare the strain-dependent interactions with host organism. Our results clearly demonstrate that: (i) MHV-68, MHV-72 and MHV-4556 differentially interact with intracellular signaling and dysregulate IFN signal transduction; (ii) MHV-68, MHV-72 and MHV-4556 degrade type I IFN receptor in very early stages of infection (2-4hpi), but not type III IFN receptor; (iii) type III IFN signaling might play a key role in antiviral defense of epithelial cells in early stages of murine gammaherpesvirus replication; (iv) NMuMG cells are an appropriate model for study of not only type I IFN signaling, but also type III IFN signaling pathway. These findings are important for better understanding of individual virus-host interactions in lytic as well as in persistent gammaherpesvirus replication and help us to elucidate IFN-III function in early events of virus infection.