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

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

Med23 inhibits HSV-1 by inducing a Type III interferon (IFN-λ) response.

<p>(a) Med23 depletion or over-expression has no effect in A549-V cells deficient in Jak/Stat signalling. A549 cells and derivative A549-V cells were transfected with Med23 siRNA SMARTpool (Med23 KD) or a pCR3-Med23 overexpression plasmid (Med23+) 48 h (siRNA) or 24 h (pCR3) before infection with HSV-1-eGFP C12. Replication was monitored, and slopes calculated and normalized to controls (RSCF siRNA or pCR3). Error bars represent the mean of at least three independent experiments. (b) Pre-treatment with Type III interferons prevents Med23-mediated enhancement of HSV-1 replication. A549 cells were mock-transfected or transfected with Med23 siRNA. After 48 h cells were untreated or pre-treated with 50 ng/ml IFN-α, IFN-β, IFN-γ or 100 ng/ml IFN-λ1 or IFN-λ2/3 for 6 h, before infecting with HSV-1-eGFP C12. Replication was monitored and slopes calculated and normalized to unstimulated, mock-transfected cells. (c) Overexpression of Med23 preferentially induces type III interferons. pCR3 or Med23 were overexpressed in A549 cells and induction of type I (IFN-β) and type III (IFN-λ1, IFN-λ2/3) was measured by qRT-PCR. mRNA levels were normalized to HPRT and calibrated to mock transfected cells (control). Error bars represent the mean of technical replicates and is representative of multiple experiments. * = <i>p</i>-value 0.003; ** = <i>p</i>-value 0.002 (unpaired t-tests for unequal variances). (d) Overexpression of Med23 induces IFN-λ secretion. A range of cell types were transfected with pCR3 or Med23, supernatant harvested 120 h post-transfection and IFN-λ3 levels measured by ELISA. Chart shows the mean and standard deviation of duplicates over two experiments. (e) siRNA depletion of Med23 inhibits the induction of IFN-λ2/3 following HSV-1 infection. A549 cells were transfected with control (RSCF) or Med23-specific siRNA (Med23 KD) before infecting with HSV-1-eGFP C12 (MOI 0.5). RNA was harvested 0 or 8 h post-infection and IFN-λ2/3 mRNA levels measured by qRT-PCR. Expression was normalized as above, and calibrated to RSCF-transfected cells at 0 h post-infection. Error bars represent the mean of technical replicates and is representative of multiple experiments.</p

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

Association of the IL28B genetic polymorphism rs12979860 with recurrence and severity of oral HSV-1 labialis.

a<p>SN, HSV-1 IgG seronegative; NR, non-recurrent; L, low recurrence; H, high recurrence; H+, high recurrence and clinical severity. See Methods for details.</p>b<p>n, number of patients.</p>c<p>M, male; F, female.</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

Occurrence and severity of clinical HSV-1 reactivation is associated with IFN-λ expression.

<p>The frequency of genotype of the single nucleotide polymorphism rs12979860 in the IFN-λ3 promoter region in HSV-1 individuals with a range of frequency and severity oral herpes labialis was determined by PCR. NR, non-recurrent; L, low recurrence; H, high recurrence; H+ high and clinically severe recurrence. Significance of genotype association was determined by Fisher's exact test, comparing the frequency of the CC, CT or TT genotype in the NR vs the L, H or H+ clinical groups. * = <i>p</i>-value 0.014. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003514#ppat-1003514-t001" target="_blank">Table 1</a> and Methods for further details.</p