Susceptibility to tuberculosis is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration

Human genetic factors predispose to tuberculosis (TB). We studied 7.6 million genetic variants in 5,530 people with pulmonary TB and in 5,607 healthy controls. In the combined analysis of these subjects and the follow-up cohort (15,087 TB patients and controls altogether), we found an association between TB and variants located in introns of the ASAP1 gene on chromosome 8q24 (P = 2.6 × 10−11 for rs4733781; P = 1.0 × 10−10 for rs10956514). Dendritic cells (DCs) showed high ASAP1 expression that was reduced after Mycobacterium tuberculosis infection, and rs10956514 was associated with the level of reduction of ASAP1 expression. The ASAP1 protein is involved in actin and membrane remodeling and has been associated with podosomes. The ASAP1-depleted DCs showed impaired matrix degradation and migration. Therefore, genetically determined excessive reduction of ASAP1 expression in M. tuberculosis–infected DCs may lead to their impaired migration, suggesting a potential mechanism of predisposition to TB

A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome

Small interfering RNAs (siRNA) genome-scale screen data for investigating host factors (HFs) involved in Herpes Simplex Virus Type 1 Replication

Small interfering RNA (siRNA) data associated with the following manuscript by Griffiths et al.(2013): A Systematic Analysis of Host Factors Reveals a Med23-Interferon-l Regulatory Axis against Herpes Simplex Virus Type 1 Replication. Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Hela cells were reverse-transfected with siRNA SMARTpools (4 siRNAs per gene). After 48 h the siRNAs were tested for cytotoxicity (3 replicates) or the capacity to influence replication of the HSV-1 GFP reporter virus C12 (6 replicates) from 24 to 80 h post-infection. Virus replication slopes during the linear phase were calculated and normalized to mock-transfected cells. Replication slopes were then compared to replication upon knockdown of essential (ICP4, VP16) or non-essential (VP11/12) viral genes, a cellular receptor for HSV-1 (HVEM) or control RISC-free siRNA (RSCF).Griffiths, Samantha; Koegl, Manfred; Boutell, Chris; Zenner, Helen; Crump, Colin; Pica, Francesca; Gonzalez, Orland; Friedel, Caroline; Barry, Gerald; Martin, Kim; Craigon, Marie; Chen, Rui; Kaza, Lakshmi; Fossum, Even; Fazakerley, John; Efstathiou, Stacey; Volpi, Antonio; Zimmer, Ralf; Ghazal, Peter; Haas, Jurgen. (2016). Small interfering RNAs (siRNA) genome-scale screen data for investigating host factors (HFs) involved in Herpes Simplex Virus Type 1 Replication, 2012-2013 [dataset]. University of Edinburgh. Medical school. Division of infection & Pathway Medicine. http://dx.doi.org/10.7488/ds/1451

Report of Regional Coordination Meeting for the North Atlantic (RCM NA) 2012

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Med23 is a novel anti-viral HF against HSV-1.

<p>(a) Multiprotein complexes involved in HSV-1 replication. The distribution of replication slopes of components from eight human protein complexes found to be critical to HSV-1 infection were ranked inhibiting (Rank 1, left) to enhancing (Rank 7157, right). Arrow denotes Med23. (b) Deconvoluted siRNAs confirm role of Mediator subunits in HSV-1 replication. The effect of four individual siRNAs (1–4) and a reconstituted SMARTpool (SP) on a range of subunits of the Mediator complex were compared to the primary screen (P). Med25 was not present in the primary screen so has no comparative ‘P’. Hela cells were transfected and infected with HSV-1-eGFP. Replication slopes were calculated and normalized to controls. Error bars represent the mean of three independent experiments done in duplicate. (c) Fluorescence microscopy and FACS analysis of Hela cells depleted of Med23. Hela cells transfected with either ICP4 or Med23 siRNA SMARTpools were infected with the recombinant HSV-1 GFP reporter virus C12 (MOI 1) and analysed by fluorescence microscopy and FACS analysis. Numbers indicate the percentage of cells in the uninfected, GFP^lo or GFP^hi populations. (d) Overexpression of Med23 inhibits HSV-1 C12-GFP and VP26-YFP. Hela or HEK cells overexpressing Med23 transiently (Hela) or stably (HEK) were infected with HSV-1 C12-eGFP or HSV-1 VP26-YFP at MOI 0.5. Replication slopes were monitored and normalized to control (pCR3)-transfected cells. Error bars represent the mean of at least three independent experiments. (e) Depletion of Med23 specifically affects HSV-1. Hela cells were depleted for Med23 with a siRNA SMARTPool and infected with HSV-1, Varicella zoster virus (VZV), human cytomegalovirus (hCMV), Vaccinia virus (VacV) or Semliki Forest Virus (SFV). Replication slopes (HSV-1, VZV, hCMV, VacV) or endpoint replication values (SFV) were calculated and normalized to controls. Error bars represent the mean of at least three independent experiments.</p

Primary validation of HFs for HSV-1.

<p>(a) Overlap between HSV-1 HFs, cellular protein interactors of HSV-1 proteins identified by Y2H system, and published protein interactors of all human herpesviral proteins. (b) Validation of a subset of HFs by siRNA deconvolution. A subset of HFs was selected for validation with deconvoluted siRNAs to confirm the phenotype observed in the primary screen. The effect of the four individual siRNAs (1–4) and a reconstituted SMARTpool (SP) were tested by reverse-transfecting into Hela cells before infecting after 48 h with HSV-1-eGFP (C12) and monitoring replication. Replication slopes were calculated and normalized as described, and compared to the primary screen slope (1°). A heat map of replication slopes was generated where red represents inhibition (replication slope <0.5) and green represents enhancement (slope >1). The phenotype was considered validated if ≥2 siRNAs produced the same or better phenotype as the primary screen. (c) Virus specificity of HSV-1 HFs. The effect of HF siRNA SMARTpools on the replication of VZV (α-herpesvirus), hCMV (β-herpesvirus) or Semliki Forest virus (SFV; RNA virus) was determined and compared to HSV-1. Normalized replication ±2×STDEV of the controls was considered inhibiting/enhancing.</p

Med23 induces IFN-λ by interacting with the transcription factor IRF7.

<p>(a) Med23 directly interacts with IRFs. Med23 was overexpressed in HEK cells as a myc-tagged fusion protein individually with a range of HA-tagged IRFs. Protein amounts were quantified and equal amounts (325 µg) were immunoprecipitated (IP) with anti-HA or anti-myc antibody before western blot analysis and staining with anti-HA (WB) to confirm protein expression (IP; anti-HA IP) and identify interaction partners (Co-IP; anti-myc IP) (b) Med23 synergistically induces IRF7-responsive promoters. A549 cells were transfected with IFN-λ1-, IFN-β- or ISRE-responsive luciferase reporter constructs with IRF7 alone or in addition to Med23. Promoter activity was determined by measurement of Firefly luciferase activity 33 hr post-transfection, and normalized to Renilla luciferase and pCR3-transfected cells. Error bars represent the mean of at least three independent experiments. Statistical significance of the synergistic increase in IFN-λ induction by Med23 with IRF7 over IFN-β induction was determined by unpaired t-tests for unequal variances. * = <i>p-value</i> 0.02.</p