Nicholas J Matheson

Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef

Summary Critical cell surface immunoreceptors downregulated during HIV infection have previously been identified using non-systematic, candidate approaches. To gain a comprehensive, unbiased overview of how HIV infection remodels the T cell surface, we took a distinct, systems-level, quantitative proteomic approach. >100 plasma membrane proteins, many without characterized immune functions, were downregulated during HIV infection. Host factors targeted by the viral accessory proteins Vpu or Nef included the amino acid transporter SNAT1 and the serine carriers SERINC3/5. We focused on SNAT1, a β-TrCP-dependent Vpu substrate. SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS. We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis. Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism.

Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells

Keeping quiet one gene at a time Chromosomal DNA comes in two flavors—euchromatin, which contains most of the expressed genes, and heterochromatin, which usually remains quiet. But what keeps genes within heterochromatin silent? Tchasovnikarova et al. examined the basis for this type of silencing in mammalian cells (see the Perspective by Brummelkamp). They identified a complex of proteins in human cells they called HUSH that kept particular parts of the genome silent by changing associated histone methylation marks. Science, this issue p. 1481, see also p. 1433 A haploid genetic screen in human cells identifies an epigenetic silencing complex that regulates heterochromatin formation. [Also see Perspective by Brummelkamp] Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.

Prevention and medical management of Clostridium difficile infection

#### Summary points The incidence of Clostridium difficile infection in the United Kingdom has increased since the late 1990s.1 High profile outbreaks in the United States, Canada, and northern Europe have been associated with a previously uncommon but highly virulent strain known as ribotype 027. A recent review in the BMJ examined the role of surgery in treating C difficile colitis.2 This review focuses on the prevention and medical management of C difficile infection. Because few randomised controlled trials (RCTs) exist on this subject, our recommendations are based mainly on non-RCT studies and clinical governance reports. #### Sources and selection criteria We searched PubMed and Google Scholar for articles published from 2006 to 2009 on the treatment and prevention of Clostridium difficile infection and screened the reference lists of retrieved publications. We also consulted the Cochrane Library and the recent best practice guidance from the Health Protection Agency . 1 C difficile can be cultured from the stool of 3% of healthy adults and as many as 35% of hospital inpatients.3 Lower rates of nosocomial colonisation are seen in some studies, and may be dependent on patient population, length of hospital stay, and local infection control procedures.w1 w2 Most colonised people remain asymptomatic. Clinical disease develops when the normal gut flora is disrupted, usually by antibiotic exposure, thereby creating conditions that favour C difficile proliferation within the colon. Although C difficile infections in England have …

Ten years experience of Salmonella infections in Cambridge, UK

OBJECTIVES Review of all Salmonella infections diagnosed in the Cambridge area over 10 years. METHODS All Salmonella enterica isolated in the Clinical Microbiology Laboratory, Addenbrookes Hospital between 1.1.1999 and 31.12.2008 were included. Patient demographics, serotype and additional relevant details (travel history, resistance-type, phage-type) were recorded. RESULTS 1003 episodes of Salmonella gastroenteritis were confirmed by stool culture, representing 88 serotypes. Serotypes Enteritidis (59%), Typhimurium (4.7%), Virchow (2.6%), Newport (1.8%) and Braenderup (1.7%) were the 5 most common isolates. There were an additional 37 invasive Salmonella infections (32 blood cultures, 4 tissue samples, 1 CSF). 13/15 patients with Salmonella Typhi or Salmonella Paratyphi isolated from blood or faeces with an available travel history had returned from the Indian subcontinent. 8/10 S. Typhi or Paratyphi isolates tested had reduced susceptibility to fluoroquinolones (MIC > or = 0.125 mg/L). 7/21 patients with non-typhoidal Salmonella bacteraemia were known to be immunosuppressed. CONCLUSION This study describes Salmonella serotypes circulating within a defined geographical area over a decade. Prospective molecular analysis of isolates of S. enterica by multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) detection will determine the geo-phylogenetic relationship of isolates within our region.

Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001

Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

Antagonism of aminoacid transport in primary CD4 T cells by HIV-1 Vpu.

BACKGROUND Less than 100 years since it was first transmitted to the human population HIV-1 infects more than 30 million people worldwide and causes almost 2 million AIDS-related deaths every year. Viruses manipulate cellular genes and pathways to benefit their survival, and the study of cell surface proteins downregulated by viruses has provided insights into both viral pathogenesis and crucial aspects of cell biology. We aimed to identify novel cell surface proteins targeted for downregulation by HIV-1. METHODS We combined plasma membrane enrichment through selective aminooxybiotinylation with tandem mass tag-based quantitative proteomics (plasma membrane profiling) to map expression timecourses of more than 800 plasma membrane proteins in T cells infected in vitro with HIV-1. Novel substrates of the viral accessory proteins Vpu and Nef were sought by use of deletion viruses and single gene overexpression. FINDINGS Our proteomic datasets defined more than 100 previously unsuspected cell surface targets of HIV-1, particularly proteins involved in T-cell activation, cell adhesion, and aminoacid transport. In addition to its known targets, Vpu was found to be necessary and sufficient for the downregulation of the aminoacid transporter TOV3. Downregulation of TOV3 was post transcriptional, mediated by the β-TrCP ubiquitin E3 ligase and occurred via an endolysosomal pathway. TOV3 was highly expressed in primary human CD4 T cells, and depletion of TOV3 by RNA interference markedly impaired the mitogenic response to CD3/CD28 stimulation. We identified alanine as an endogenous TOV3 substrate, and showed that extracellular alanine was crucial for T-cell proliferation. INTERPRETATION This study suggests that TOV3 downregulation is restricted to Vpu variants from the lineage of HIV-1 group M viruses giving rise to pandemic AIDS. Antagonism of alanine transport in CD4 T cells might contribute to HIV-1 pathogenesis through modulation of virus production, impairment of the adaptive immune response, or enhancement of CD4 T-cell loss. FUNDING Wellcome Trust, Addenbrookes Charitable Trust, Raymond and Beverly Sackler Foundation.

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

Viruses manipulate host cells to enhance their replication, and the identification of host factors targeted by viruses has led to key insights in both viral pathogenesis and cellular physiology. We previously described global changes in cellular protein levels during human immunodeficiency virus (HIV) infection using transformed CEM-T4 T cells as a model. In this study, we develop an HIV reporter virus 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, including quantitation of >9,000 proteins across 4 different donors, and temporal profiling during T cell activation. Remarkably, amongst 650 cellular proteins significantly perturbed during HIV infection of primary T cells (q<0.05), almost 50% are regulated directly or indirectly by the viral accessory proteins Vpr, Vif, Nef and Vpu. The remainder have not been previously characterised, but include novel Vif-dependent targets FMR1 and DPH7, and 192 targets not identified and/or regulated in T cell lines, such as AIRD5A and PTPN22. We therefore provide a high-coverage functional proteomic atlas of HIV infection, and a mechanistic account of HIV-dependent changes in its natural target cell.

Vpr drives massive cellular proteome remodelling in HIV-1 infection

Abstract 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 characterised cellular targets have been associated with Vif, Vpu and Nef, which counteract host restriction and promote viral replication. Conversely, whilst 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 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.Viral infection causes global remodelling of the cellular proteome. We previously mapped the temporal changes in abundance of thousands of proteins in HIV-1 infected cells (Greenwood & Matheson, 2016). While a small proportion of these changes were attributable to specific HIV-1 accessory proteins, most were unexplained. Here, we use complementary unbiased mass spectrometry-based approaches to demonstrate that a single viral protein, Vpr, is both necessary and sufficient to cause the vast majority of these changes. This global protein regulation requires substrate binding and degradation via DCAF1, but is mostly independent of Vpr-mediated cell cycle arrest. Combined approaches of pulsed-Stable Isotope Labelling with Amino Acids in Cell Culture (pulsed-SILAC) and immunoprecipitation-mass spectrometry (IP-MS) identified at least 38 such cellular proteins directly targeted for degradation by Vpr. Thus, whilst other HIV-1 accessory proteins downregulate a small number of host factors, Vpr degrades multiple protein targets, causing systems-level remodelling of the cellular proteome. Impact statement HIV infection causes massive changes to the cellular proteome and a single HIV protein, Vpr, is necessary and sufficient to drive almost all these changes by degrading multiple host proteins.

Plasma membrane proteomics identifies Notch1 as a potential regulator of ras-induced senescence

Abstract Background Oncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism leading to stable cell-cycle arrest in response to oncogene activation. OIS is a heterogeneous phenotype of multiple effector mechanisms; understanding of in-vivo OIS is lacking because of the difficulty in identifying senescence. We wished to establish a cell-surface phenotype of OIS. Methods We used ER:Ras IMR90 human diploid fibroblasts, which undergo OIS after 6 days with 4-hydroxytamoxifen (4-OHT). We used SILAC (stable isotope labelling with amino acids in cell culture)-based proteomics and three labelling conditions (light, IMR90 plus 4-OHT; medium [lysine, K+4Da, arginine, R+6Da] ER:ras IMR90; heavy [K+8Da, R+10Da] ER:Ras IMR90 plus 4-OHT), combined with cell surface aminooxybiotinylation before streptavidin pulldown. Tryptic peptides were fractionated by high pH reversed-phase high performance liquid chromatogrpahy and then subjected to liquid chromatograph mass spectrometry. Data were processed by the analytical packages MaxQuant and MASCOT. Hits were validated by fluorescence-activated cell sorting (FACS), quantitative PCR, and immunoblotting. shRNA-mediated knockdown of protein expression used the murine stem cell virus-miR30 system. Findings 899 proteins were identified, of which 73% were present at the cell surface by Gene Ontology annotation. Notch1 was significantly upregulated in OIS compared with both control conditions (3·1–3·4 fold). Upregulation was confirmed by both FACS and immunoblotting. Downstream Notch target-genes were also upregulated in OIS. Treatment with the γ-secretase inhibitor DAPT increased both senescence-associated heterochromatin foci (SAHF) and senescence-associated β galactosidase (SA-β-gal) activity, features of OIS. However, Notch1 knockdown reduced both SAHF and SA-β-gal. The effect of DAPT was not mediated through canonical Notch1 signalling since RBP-j knockdown had no effect upon DAPT-mediated SAHF and SA β-gal upregulation. Numb, a canonical Notch pathway inhibitor, is upregulated in OIS. Numb may underpin a transition switch from canonical to non-canonical Notch signaling in OIS. Interpretation Plasma membrane proteomics has identified a cell-surface phenotype of OIS. Notch 1 cell surface expression and downstream target-genes are upregulated in OIS. The transition to OIS is correlated with a transition from canonical to non-canonical Notch1 signalling that may be driven by Numb expression. Funding Cancer Research UK.