Publications by authors named "Paul J Lehner"

115 Publications

Human embryonic stem cell-derived cardiomyocyte platform screens inhibitors of SARS-CoV-2 infection.

Commun Biol 2021 07 29;4(1):926. Epub 2021 Jul 29.

Experimental Medicine and Immunotherapeutics, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated.
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http://dx.doi.org/10.1038/s42003-021-02453-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322398PMC
July 2021

CD97 stabilises the immunological synapse between dendritic cells and T cells and is targeted for degradation by the Salmonella effector SteD.

PLoS Pathog 2021 Jul 27;17(7):e1009771. Epub 2021 Jul 27.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.

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. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.
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http://dx.doi.org/10.1371/journal.ppat.1009771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345877PMC
July 2021

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

Immunity 2021 06 16;54(6):1257-1275.e8. Epub 2021 May 16.

R&D Department, Hycult Biotech, 5405 PD Uden, the Netherlands.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8 T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.
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http://dx.doi.org/10.1016/j.immuni.2021.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125900PMC
June 2021

Single-dose BNT162b2 vaccine protects against asymptomatic SARS-CoV-2 infection.

Elife 2021 04 8;10. Epub 2021 Apr 8.

Cambridge University NHS Hospitals Foundation Trust, Cambridge, United Kingdom.

The BNT162b2 mRNA COVID-19 vaccine (Pfizer-BioNTech) is being utilised internationally for mass COVID-19 vaccination. Evidence of single-dose protection against symptomatic disease has encouraged some countries to opt for delayed booster doses of BNT162b2, but the effect of this strategy on rates of asymptomatic SARS-CoV-2 infection remains unknown. We previously demonstrated frequent pauci- and asymptomatic SARS-CoV-2 infection amongst healthcare workers (HCWs) during the UK's first wave of the COVID-19 pandemic, using a comprehensive PCR-based HCW screening programme (Rivett et al., 2020; Jones et al., 2020). Here, we evaluate the effect of first-dose BNT162b2 vaccination on test positivity rates and find a fourfold reduction in asymptomatic infection amongst HCWs ≥12 days post-vaccination. These data provide real-world evidence of short-term protection against asymptomatic SARS-CoV-2 infection following a single dose of BNT162b2 vaccine, suggesting that mass first-dose vaccination will reduce SARS-CoV-2 , as well as the burden of COVID-19 .
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http://dx.doi.org/10.7554/eLife.68808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064747PMC
April 2021

Neural stem cells traffic functional mitochondria via extracellular vesicles.

PLoS Biol 2021 04 7;19(4):e3001166. Epub 2021 Apr 7.

Department of Clinical Neurosciences and NIHR Biomedical Research Centre, University of Cambridge, United Kingdom.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.
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http://dx.doi.org/10.1371/journal.pbio.3001166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055036PMC
April 2021

The SMC5/6 complex compacts and silences unintegrated HIV-1 DNA and is antagonized by Vpr.

Cell Host Microbe 2021 05 2;29(5):792-805.e6. Epub 2021 Apr 2.

Cambridge Institute for Therapeutic Immunology & Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0AW, UK. Electronic address:

Silencing of nuclear DNA is an essential feature of innate immune responses to invading pathogens. Early in infection, unintegrated lentiviral cDNA accumulates in the nucleus yet remains poorly expressed. In HIV-1-like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonizes cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library and identify SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. ATAC-seq demonstrates that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting that the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines its targeting by Vpr as an evolutionarily conserved antagonism.
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http://dx.doi.org/10.1016/j.chom.2021.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118623PMC
May 2021

No evidence for basigin/CD147 as a direct SARS-CoV-2 spike binding receptor.

Sci Rep 2021 01 11;11(1):413. Epub 2021 Jan 11.

Cell Surface Signalling Laboratory, Wellcome Sanger Institute, Cambridge, UK.

The spike protein of SARS-CoV-2 is known to enable viral invasion into human cells through direct binding to host receptors including ACE2. An alternate entry receptor for the virus was recently proposed to be basigin/CD147. These early studies have already prompted a clinical trial and multiple published hypotheses speculating on the role of this host receptor in viral infection and pathogenesis. Here, we report that we are unable to find evidence supporting the role of basigin as a putative spike binding receptor. Recombinant forms of the SARS-CoV-2 spike do not interact with basigin expressed on the surface of human cells, and by using specialized assays tailored to detect receptor interactions as weak or weaker than the proposed basigin-spike binding, we report no evidence for a direct interaction between the viral spike protein to either of the two common isoforms of basigin. Finally, removing basigin from the surface of human lung epithelial cells by CRISPR/Cas9 results in no change in their susceptibility to SARS-CoV-2 infection. Given the pressing need for clarity on which viral targets may lead to promising therapeutics, we present these findings to allow more informed decisions about the translational relevance of this putative mechanism in the race to understand and treat COVID-19.
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http://dx.doi.org/10.1038/s41598-020-80464-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801465PMC
January 2021

Treatment of COVID-19 with remdesivir in the absence of humoral immunity: a case report.

Nat Commun 2020 12 14;11(1):6385. Epub 2020 Dec 14.

Belfast Health and Social Care Trust, Belfast, Northern Ireland, UK.

The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. This unusual clinical course is consistent with a contribution of antibodies to both viral clearance and progression to severe disease. In the absence of these confounders, we take an experimental medicine approach to examine the in vivo utility of remdesivir. Over two independent courses of treatment, we observe a temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients.
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http://dx.doi.org/10.1038/s41467-020-19761-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736571PMC
December 2020

Dissecting Herpes Simplex Virus 1-Induced Host Shutoff at the RNA Level.

J Virol 2021 01 13;95(3). Epub 2021 Jan 13.

Institute for Virology and Immunobiology, Julius-Maximilians-Universität Würzburg, Würzburg, Germany

Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff () protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δ mutant infection of primary human fibroblasts. Following virus entry, activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δ infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed -nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a -dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection. The HSV-1 virion host shutoff () protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified -dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to -mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes.
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http://dx.doi.org/10.1128/JVI.01399-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925104PMC
January 2021

A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export.

Nat Commun 2020 11 3;11(1):5559. Epub 2020 Nov 3.

Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, UK.

Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.
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http://dx.doi.org/10.1038/s41467-020-19032-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642327PMC
November 2020

Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation.

Cell Rep 2020 10;33(2):108249

Department of Medicine, University of Cambridge, Hills Road, Cambridge CB2 0QQ, UK; Cambridge Institute for Therapeutic Immunology and Infectious Disease (CITIID), University of Cambridge, Puddicombe Way, Cambridge CB2 0AW, UK. Electronic address:

Kaposi's sarcoma herpesvirus (KSHV) is an oncogenic human virus and the leading cause of mortality in HIV infection. KSHV reactivation from latent- to lytic-stage infection initiates a cascade of viral gene expression. Here we show how these changes remodel the host cell proteome to enable viral replication. By undertaking a systematic and unbiased analysis of changes to the endothelial cell proteome following KSHV reactivation, we quantify >7,000 cellular proteins and 71 viral proteins and provide a temporal profile of protein changes during the course of lytic KSHV infection. Lytic KSHV induces >2-fold downregulation of 291 cellular proteins, including PKR, the key cellular sensor of double-stranded RNA. Despite the multiple episomes per cell, CRISPR-Cas9 efficiently targets KSHV genomes. A complementary KSHV genome-wide CRISPR genetic screen identifies K5 as the viral gene responsible for the downregulation of two KSHV targets, Nectin-2 and CD155, ligands of the NK cell DNAM-1 receptor.
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http://dx.doi.org/10.1016/j.celrep.2020.108249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567700PMC
October 2020

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control.

Nat Commun 2020 10 2;11(1):4940. Epub 2020 Oct 2.

Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK.

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.
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http://dx.doi.org/10.1038/s41467-020-18761-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532188PMC
October 2020

Periphilin self-association underpins epigenetic silencing by the HUSH complex.

Nucleic Acids Res 2020 10;48(18):10313-10328

Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.
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http://dx.doi.org/10.1093/nar/gkaa785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544229PMC
October 2020

Point of Care Nucleic Acid Testing for SARS-CoV-2 in Hospitalized Patients: A Clinical Validation Trial and Implementation Study.

Cell Rep Med 2020 Aug 15;1(5):100062. Epub 2020 Jul 15.

Department of Infectious Diseases, Cambridge University NHS Hospitals Foundation Trust, Cambridge CB2 0QQ, UK.

There is an urgent need for rapid SARS-CoV-2 testing in hospitals to limit nosocomial spread. We report an evaluation of point of care (POC) nucleic acid amplification testing (NAAT) in 149 participants with parallel combined nasal and throat swabbing for POC versus standard lab RT-PCR testing. Median time to result is 2.6 (IQR 2.3-4.8) versus 26.4 h (IQR 21.4-31.4, p < 0.001), with 32 (21.5%) positive and 117 (78.5%) negative. Cohen's κ correlation between tests is 0.96 (95% CI 0.91-1.00). When comparing nearly 1,000 tests pre- and post-implementation, the median time to definitive bed placement from admission is 23.4 (8.6-41.9) versus 17.1 h (9.0-28.8), p = 0.02. Mean length of stay on COVID-19 "holding" wards is 58.5 versus 29.9 h (p < 0.001). POC testing increases isolation room availability, avoids bed closures, allows discharge to care homes, and expedites access to hospital procedures. POC testing could mitigate the impact of COVID-19 on hospital systems.
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http://dx.doi.org/10.1016/j.xcrm.2020.100062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362826PMC
August 2020

How does SARS-CoV-2 cause COVID-19?

Science 2020 07;369(6503):510-511

Department of Medicine, Cambridge Institute for Therapeutic Immunology and Infectious Disease (CITIID), University of Cambridge, Cambridge, UK.

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http://dx.doi.org/10.1126/science.abc6156DOI Listing
July 2020

Ubiquitin-mediated regulation of sterol homeostasis.

Curr Opin Cell Biol 2020 08 21;65:103-111. Epub 2020 Jun 21.

Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, United Kingdom. Electronic address:

Cholesterol is an essential component of mammalian membranes, and its homeostasis is strictly regulated, with imbalances causing atherosclerosis, Niemann Pick disease, and familial hypercholesterolemia. Cellular cholesterol supply is mediated by LDL-cholesterol import and de novo cholesterol biosynthesis, and both pathways are adjusted to cellular demand by the cholesterol-sensitive SREBP2 transcription factor. Cholesterol homeostasis is modulated by a wide variety of metabolic pathways and the ubiquitination machinery, in particular E3 ubiquitin ligases. In this article, we review recent progress in understanding the role of E3 ubiquitin ligases in the metabolic control of cellular sterol homeostasis.
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http://dx.doi.org/10.1016/j.ceb.2020.04.010DOI Listing
August 2020

Effective control of SARS-CoV-2 transmission between healthcare workers during a period of diminished community prevalence of COVID-19.

Elife 2020 06 19;9. Epub 2020 Jun 19.

Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

Previously, we showed that 3% (31/1032)of asymptomatic healthcare workers (HCWs) from a large teaching hospital in Cambridge, UK, tested positive for SARS-CoV-2 in April 2020. About 15% (26/169) HCWs with symptoms of coronavirus disease 2019 (COVID-19) also tested positive for SARS-CoV-2 (Rivett et al., 2020). Here, we show that the proportion of both asymptomatic and symptomatic HCWs testing positive for SARS-CoV-2 rapidly declined to near-zero between 25th April and 24th May 2020, corresponding to a decline in patient admissions with COVID-19 during the ongoing UK 'lockdown'. These data demonstrate how infection prevention and control measures including staff testing may help prevent hospitals from becoming independent 'hubs' of SARS-CoV-2 transmission, and illustrate how, with appropriate precautions, organizations in other sectors may be able to resume on-site work safely.
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http://dx.doi.org/10.7554/eLife.59391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326489PMC
June 2020

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission.

Elife 2020 05 11;9. Epub 2020 May 11.

Department of Infectious Diseases, Cambridge University NHS Hospitals Foundation Trust, Cambridge, United Kingdom.

Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3 week period (April 2020), 1032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19)>7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B∙1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff.
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http://dx.doi.org/10.7554/eLife.58728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314537PMC
May 2020

Integrative functional genomics decodes herpes simplex virus 1.

Nat Commun 2020 04 27;11(1):2038. Epub 2020 Apr 27.

Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Versbacher Straße 7, 97078, Würzburg, Germany.

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.
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http://dx.doi.org/10.1038/s41467-020-15992-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184758PMC
April 2020

Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest.

Elife 2020 04 15;9. Epub 2020 Apr 15.

Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

The seminal description of the cellular restriction factor APOBEC3G and its antagonism by HIV-1 Vif has underpinned two decades of research on the host-virus interaction. We recently reported that HIV-1 Vif is also able to degrade the PPP2R5 family of regulatory subunits of key cellular phosphatase PP2A (PPP2R5A-E; Greenwood et al., 2016; Naamati et al., 2019). We now identify amino acid polymorphisms at positions 31 and 128 of HIV-1 Vif which selectively regulate the degradation of PPP2R5 family proteins. These residues covary across HIV-1 viruses in vivo, favouring depletion of PPP2R5A-E. Through analysis of point mutants and naturally occurring Vif variants, we further show that degradation of PPP2R5 family subunits is both necessary and sufficient for Vif-dependent G2/M cell cycle arrest. Antagonism of PP2A by HIV-1 Vif is therefore independent of APOBEC3 family proteins, and regulates cell cycle progression in HIV-infected cells.
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http://dx.doi.org/10.7554/eLife.53036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920553PMC
April 2020

A genome-wide CRISPR screen identifies regulation factors of the TLR3 signalling pathway.

Innate Immun 2020 08 4;26(6):459-472. Epub 2020 Apr 4.

Institut Curie, PSL Research University, INSERM U932, France.

A subset of TLRs is specialised in the detection of incoming pathogens by sampling endosomes for nucleic acid contents. Among them, TLR3 senses the abnormal presence of double-stranded RNA in the endosomes and initiates a potent innate immune response via activation of NF-κB and IRF3. Nevertheless, mechanisms governing TLR3 regulation remain poorly defined. To identify new molecular players involved in the TLR3 pathway, we performed a genome-wide screen using CRISPR/Cas9 technology. We generated TLR3 reporter cells carrying a NF-κB-responsive promoter that controls GFP expression. Cells were next transduced with a single-guide RNA (sgRNA) library, subjected to sequential rounds of stimulation with poly(I:C) and sorting of the GFP-negative cells. Enrichments in sgRNA estimated by deep sequencing identified genes required for TLR3-induced activation of NF-κB. Among the hits, five genes known to be critically involved in the TLR3 pathway, including TLR3 itself and the chaperone UNC93B1, were identified by the screen, thus validating our strategy. We further studied the top 40 hits and focused on the transcription factor aryl hydrocarbon receptor (AhR). Depletion of AhR had a dual effect on the TLR3 response, abrogating IL-8 production and enhancing IP-10 release. Moreover, in primary human macrophages exposed to poly(I:C), AhR activation enhanced IL-8 and diminished IP-10 release. Overall, these results reveal AhR plays a role in the TLR3 cellular innate immune response.
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http://dx.doi.org/10.1177/1753425920915507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491238PMC
August 2020

Stromal cell protein kinase C-β inhibition enhances chemosensitivity in B cell malignancies and overcomes drug resistance.

Sci Transl Med 2020 01;12(526)

Wellcome Trust/MRC Cambridge Stem Cell Institute and Department of Haematology, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AH, UK.

Overcoming drug resistance remains a key challenge to cure patients with acute and chronic B cell malignancies. Here, we describe a stromal cell-autonomous signaling pathway, which contributes to drug resistance of malignant B cells. We show that protein kinase C (PKC)-β-dependent signals from bone marrow-derived stromal cells markedly decrease the efficacy of cytotoxic therapies. Conversely, small-molecule PKC-β inhibitors antagonize prosurvival signals from stromal cells and sensitize tumor cells to targeted and nontargeted chemotherapy, resulting in enhanced cytotoxicity and prolonged survival in vivo. Mechanistically, stromal PKC-β controls the expression of adhesion and matrix proteins, required for activation of phosphoinositide 3-kinases (PI3Ks) and the extracellular signal-regulated kinase (ERK)-mediated stabilization of B cell lymphoma-extra large (BCL-X) in tumor cells. Central to the stroma-mediated drug resistance is the PKC-β-dependent activation of transcription factor EB, regulating lysosome biogenesis and plasma membrane integrity. Stroma-directed therapies, enabled by direct inhibition of PKC-β, enhance the effectiveness of many antileukemic therapies.
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http://dx.doi.org/10.1126/scitranslmed.aax9340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116365PMC
January 2020

Interferon-Responsive Genes Are Targeted during the Establishment of Human Cytomegalovirus Latency.

mBio 2019 12 3;10(6). Epub 2019 Dec 3.

Department of Medicine, University of Cambridge, Cambridge, United Kingdom

Human cytomegalovirus (HCMV) latency is an active process which remodels the latently infected cell to optimize latent carriage and reactivation. This is achieved, in part, through the expression of viral genes, including the G-protein-coupled receptor US28. Here, we use an unbiased proteomic screen to assess changes in host proteins induced by US28, revealing that interferon-inducible genes are downregulated by US28. We validate that major histocompatibility complex (MHC) class II and two pyrin and HIN domain (PYHIN) proteins, myeloid cell nuclear differentiation antigen (MNDA) and IFI16, are downregulated during experimental latency in primary human CD14 monocytes. We find that IFI16 is targeted rapidly during the establishment of latency in a US28-dependent manner but only in undifferentiated myeloid cells, a natural site of latent carriage. Finally, by overexpressing IFI16, we show that IFI16 can activate the viral major immediate early promoter and immediate early gene expression during latency via NF-κB, a function which explains why downregulation of IFI16 during latency is advantageous for the virus. Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which infects 50 to 100% of humans worldwide. HCMV causes a lifelong subclinical infection in immunocompetent individuals but is a serious cause of mortality and morbidity in the immunocompromised and neonates. In particular, reactivation of HCMV in the transplant setting is a major cause of transplant failure and related disease. Therefore, a molecular understanding of HCMV latency and reactivation could provide insights into potential ways to target the latent viral reservoir in at-risk patient populations.
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http://dx.doi.org/10.1128/mBio.02574-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890990PMC
December 2019

An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer.

Cancer Cell 2019 10 26;36(4):385-401.e8. Epub 2019 Sep 26.

Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3052, Australia; Centre for Cancer Research, University of Melbourne, Parkville, Australia. Electronic address:

Loss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.
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http://dx.doi.org/10.1016/j.ccell.2019.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876280PMC
October 2019

Temporal Proteomic Analysis of BK Polyomavirus Infection Reveals Virus-Induced G Arrest and Highly Effective Evasion of Innate Immune Sensing.

J Virol 2019 08 30;93(16). Epub 2019 Jul 30.

Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom

BK polyomavirus (BKPyV) is a small DNA virus that establishes a life-long persistent infection in the urinary tract of most people. BKPyV is known to cause severe morbidity in renal transplant recipients and can lead to graft rejection. The simple 5.2-kbp double-stranded DNA (dsDNA) genome expresses just seven known proteins; thus, it relies heavily on the host machinery to replicate. How the host proteome changes over the course of infection is key to understanding this host-virus interplay. Here, for the first time quantitative temporal viromics has been used to quantify global changes in >9,000 host proteins in two types of primary human epithelial cells throughout 72 h of BKPyV infection. These data demonstrate the importance of cell cycle progression and pseudo-G arrest in effective BKPyV replication, along with a surprising lack of an innate immune response throughout the whole virus replication cycle. BKPyV thus evades pathogen recognition to prevent activation of innate immune responses in a sophisticated manner. BK polyomavirus can cause serious problems in immune-suppressed patients, in particular, kidney transplant recipients who can develop polyomavirus-associated kidney disease. In this work, we have used advanced proteomics techniques to determine the changes to protein expression caused by infection of two independent primary cell types of the human urinary tract (kidney and bladder) throughout the replication cycle of this virus. Our findings have uncovered new details of a specific form of cell cycle arrest caused by this virus, and, importantly, we have identified that this virus has a remarkable ability to evade detection by host cell defense systems. In addition, our data provide an important resource for the future study of kidney epithelial cells and their infection by urinary tract pathogens.
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http://dx.doi.org/10.1128/JVI.00595-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675895PMC
August 2019

Promiscuous Targeting of Cellular Proteins by Vpr Drives Systems-Level Proteomic Remodeling in HIV-1 Infection.

Cell Rep 2019 04;27(5):1579-1596.e7

Department of Medicine, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0QQ, UK; Cambridge Institute for Medical Research, Keith Peters Building, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0XY, UK; Cambridge Institute of Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, Cambridge CB2 0AW, UK. Electronic address:

HIV-1 encodes four "accessory proteins" (Vif, Vpr, Vpu, and Nef), dispensable for viral replication in vitro but essential for viral pathogenesis in vivo. Well characterized cellular targets have been associated with Vif, Vpu, and Nef, which counteract host restriction and promote viral replication. Conversely, although several substrates of Vpr have been described, their biological significance remains unclear. Here, we use complementary unbiased mass spectrometry-based approaches to demonstrate that Vpr is both necessary and sufficient for the DCAF1/DDB1/CUL4 E3 ubiquitin ligase-mediated degradation of at least 38 cellular proteins, causing systems-level changes to the cellular proteome. We therefore propose that promiscuous targeting of multiple host factors underpins complex Vpr-dependent cellular phenotypes and validate this in the case of G2/M cell cycle arrest. Our model explains how Vpr modulates so many cell biological processes and why the functional consequences of previously described Vpr targets, identified and studied in isolation, have proved elusive.
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http://dx.doi.org/10.1016/j.celrep.2019.04.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506760PMC
April 2019

Functional proteomic atlas of HIV infection in primary human CD4+ T cells.

Elife 2019 03 12;8. Epub 2019 Mar 12.

Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

Viruses manipulate host cells to enhance their replication, and the identification of cellular factors targeted by viruses has led to key insights into both viral pathogenesis and cell biology. In this study, we develop an HIV reporter virus (HIV-AFMACS) displaying a streptavidin-binding affinity tag at the surface of infected cells, allowing facile one-step selection with streptavidin-conjugated magnetic beads. We use this system to obtain pure populations of HIV-infected primary human CD4+ T cells for detailed proteomic analysis, and quantitate approximately 9000 proteins across multiple donors on a dynamic background of T cell activation. Amongst 650 HIV-dependent changes (q < 0.05), we describe novel Vif-dependent targets FMR1 and DPH7, and 192 proteins not identified and/or regulated in T cell lines, such as ARID5A and PTPN22. We therefore provide a high-coverage functional proteomic atlas of HIV infection, and a mechanistic account of host factors subverted by the virus in its natural target cell.
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http://dx.doi.org/10.7554/eLife.41431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414203PMC
March 2019

Monocytes Latently Infected with Human Cytomegalovirus Evade Neutrophil Killing.

iScience 2019 Feb 8;12:13-26. Epub 2019 Jan 8.

Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0QQ, UK. Electronic address:

One site of latency of human cytomegalovirus (HCMV) in vivo is in undifferentiated cells of the myeloid lineage. Although latently infected cells are known to evade host T cell responses by suppression of T cell effector functions, it is not known if they must also evade surveillance by other host immune cells. Here we show that cells latently infected with HCMV can, indeed, be killed by host neutrophils but only in a serum-dependent manner. Specifically, antibodies to the viral latency-associated US28 protein mediate neutrophil killing of latently infected cells. To address this mechanistically, a full proteomic screen was carried out on latently infected monocytes. This showed that latent infection downregulates the neutrophil chemoattractants S100A8/A9, thus suppressing neutrophil recruitment to latently infected cells. The ability of latently infected cells to inhibit neutrophil recruitment represents an immune evasion strategy of this persistent human pathogen, helping to prevent clearance of the latent viral reservoir.
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http://dx.doi.org/10.1016/j.isci.2019.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6352302PMC
February 2019

An Interferon-Driven Oxysterol-Based Defense against Tumor-Derived Extracellular Vesicles.

Cancer Cell 2019 01;35(1):33-45.e6

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Tumor-derived extracellular vesicles (TEV) "educate" healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy.
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http://dx.doi.org/10.1016/j.ccell.2018.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6336114PMC
January 2019

The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.

Elife 2018 12 13;7. Epub 2018 Dec 13.

Department of Medicine, Cambridge Institute for Medical Research, Cambridge, United Kingdom.

Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.

Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
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http://dx.doi.org/10.7554/eLife.40009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292692PMC
December 2018
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