Pivotal role for the ESCRT-II complex subunit EAP30/SNF8 in IRF3-dependent innate antiviral defense

<div><p>The activation of interferon (IFN)-regulatory factor-3 (IRF3), characterized by phosphorylation and nuclear translocation of the latent transcription factor, is central to initiating innate antiviral responses. Whereas much has been learned about the upstream pathways and signaling mechanisms leading to IRF3 activation, how activated IRF3 operates in the nucleus to control transcription of IFNs remains obscure. Here we identify EAP30 (a.k.a, SNF8/VPS22), an endosomal sorting complex required for transport (ESCRT)-II subunit, as an essential factor controlling IRF3-dependent antiviral defense. Depletion of EAP30, but not other ESCRT-II subunits, compromised IRF3-dependent induction of type I and III IFNs, IFN-stimulated genes (ISGs) and chemokines by double-stranded RNA or viruses. EAP30, however, was dispensable for the induction of inflammatory mediators of strict NF-κB target. Significantly, knockdown of EAP30 also impaired the establishment of an antiviral state against vesicular stomatitis virus and hepatitis C virus, which are of distinct viral families. Mechanistically, EAP30 was not required for IRF3 activation but rather acted at a downstream step. Specifically, a fraction of EAP30 localized within the nucleus, where it formed a complex with IRF3 and its transcriptional co-activator, CREB-binding protein (CBP), in a virus-inducible manner. These interactions promoted IRF3 binding to target gene promoters such as IFN-β, IFN-λ1 and ISG56. Together, our data describe an unappreciated role for EAP30 in IRF3-dependent innate antiviral response in the nucleus.</p></div

Knockdown of EAP30 compromises cellular innate antiviral defense.

<p><b>(A)</b> PH5CH8 cells transfected with control or EAP30 siRNA for 48 h were incubated with or without poly(I:C) (20 μg/ml) for 16 h prior to challenge with VSV-Luc (MOI = 0.1) for 6 h. Luciferase assay was performed to monitor VSV replication levels. <b>(B)</b> Control siRNA- or EAP30 siRNA-transfected-Huh7-TLR3 cells were mock-treated or transfected with 2 μg of poly(I:C) for 16 h prior to HCV-JFH1 infection (MOI = 0.05) for 48 h. Intracellular HCV RNA levels (relative to 28S rRNA) were quantified by qPCR. <b>(C)</b> PH5CH8 cells transiently overexpressing control vector or EAP30 were incubated with poly(I:C) for 16 h prior to challenge with VSV-Luc (MOI = 0.1) for 6 h. Luciferase assay was performed to monitor VSV replication levels. <b>(D)</b> Control vector- or EAP30-overexpressing Huh7-TLR3 cells were mock-treated or transfected with poly(I:C) for 16 h prior to HCV-JFH1 infection (MOI = 0.05) for 48 h. Intracellular HCV RNA levels (relative to 28S rRNA) were quantified by qPCR.</p

Impact of ESCRT-II knockdown on activation of the IFN-β promoter by ectopic expression of various signaling proteins in TLR3 and RIG-I pathways.

<p><b>(A)</b> IFN-β promoter activity following ectopic expression of control vector, TRIF, TBK1, IKKε or IRF3-5D in PH5CH8 cells that had been transfected with the indicated siRNAs. <b>(B)</b> IFN-β promoter activity following ectopic expression of control vector, RIG-I CARD (RIG-I-N), MDA5, or MAVS in PH5CH8 cells that had been transfected with the indicated siRNAs.“*”, “**”, and “***” denote statistical differences exist as compared with control siRNA-transfected cells with a <i>P</i>-value of < 0.05, < 0.01 and <0.001, respectively.</p

EAP30 silencing decreases poly(I:C)- and SeV-induced ISGs expression but not ISG induction by IFN-α.

<p><b>(A)</b> qPCR analysis of OASL, PKR, MX1 and ISG15 mRNA levels in PH5CH8 cells transfected with control, IRF3, or EAP30 siRNA for 48 h, and mock-treated (empty bars)or stimulated with poly(I:C) (10 μg/ml) for additional 8 h (gray bars). <b>(B)</b> Immunoblotting of ISG56, IRF3, EAP30 and actin proteins under experimental conditions of (A). <b>(C)</b> qPCR quantification of OASL, PKR, MX1 and ISG15 mRNA levels in PH5CH8 cells transfected with control, IRF3, or EAP30 siRNA for 48 h, and mock-treated (empty bars)or infected with SeV (160 HAU/ml) for additional 8 h (solid bars). <b>(D)</b> Immunoblotting of ISG56, SeV, IRF3, EAP30 and actin proteins under experimental conditions of (C). <b>(E)</b> qPCR quantification of OASL, PKR, MX1 and ISG15 mRNA levels in PH5CH8 cells transfected with control, IRF3, or EAP30 siRNA for 48 h, and mock-treated (empty bars)or stimulated with IFN-α (100 U/ml) for additional 8 h (solid bars). “*” and “**” denote statistical differences exist as compared with control siRNA-transfected cells with a <i>P</i>-value of < 0.05 and < 0.01, respectively.</p

Knockdown of EAP30 does not affect virus-induced IRF3 phosphorylation, dimerization or nuclear translocation, but impairs IRF3-CBP complex formation and IRF3 binding to target gene promoters.

<p><b>(A)</b> Immunoblotting of phosphorylated-IRF3 (p-IRF3), SeV, EAP30 and action levels in PH5CH8 cells transfected with control or EAP30 siRNA for 48 h and mock-infected or infected with SeV for additional 8 h. <b>(B)</b> Immunoblotting of IRF3 monomer and dimer forms following native PAGE of the samples shown in (A). <b>(C)</b> Immunoblotting of IRF3, SeV, EAP30, lamin A/C (nuclear protein marker), β-tubulin (cytoplasmic protein marker), and actin loading control in cytoplasmic (CE) and nuclear (NE) fractions of PH5CH8 cells transfected with control or EAP30 siRNA and mock-infected or infected with SeV. <b>(D)</b> Immunoblotting of p-IRF3, actin, ISG56, SeV, and EAP30 in HEK293-shEAP30 and HEK293-shCon cells mock-infected or infected with SeV for 8 h. <b>(E)</b> Whole cells lysate (WCL) were collected from HEK293-shEAP30 and HEK293-shCon cells that were mock-infected or infected with SeV for immunoblotting of CBP, IRF3, EAP30 and actin (left panel) and co-IP analysis of virus-induced CBP-IRF3 association (right panel).<b>(F)</b> ChIP analysis of IRF3 binding to IFNβ, IFNL1, and IFIT1 (ISG56) promoters in nuclear extracts of HEK293-shEAP30 and HEK293-shCon cells that were mock-infected or infected with SeV. The ChIP-enriched DNA levels were analyzed by qPCR and normalized to input DNA, followed by subtraction of nonspecific binding determined using control IgG. “*” denotes statistical differences exist with a <i>P</i>-value of < 0.05.</p

Impact of ESCRT-II knockdown on dsRNA- or virus-induced chemokine expression.

<p><b>(A)</b> qPCR analysis of RANTES and IP-10 mRNA levels in PH5CH8 cells transfected with indicated siRNAs and mock-treated (empty bars), stimulated by 10 μg/ml of poly(I:C) for 6 h (grey bars), or infected with SeV at 160 HAU/ml for 8 h (black bars). <b>(B)</b> qPCR analysis of RANTES and IP-10 mRNA levels in Huh7.5-TLR3 cells transfected with indicated siRNAs and mock-treated (empty bars), stimulated by 10 μg/ml of poly(I:C) for 6 h (grey bars), or infected with HCV-JFH1 (MOI = 0.1) for 56 h (black bars). For the HCV groups cells were infected for 8 h prior to siRNA transfection for additional 48 h. “*” and “**” denote statistical differences exist as compared with control siRNA-transfected cells with a <i>P</i>-value of < 0.05 and < 0.01, respectively.</p

Knockdown of EAP30 compromises cellular innate antiviral defense.

<p><b>(A)</b> PH5CH8 cells transfected with control or EAP30 siRNA for 48 h were incubated with or without poly(I:C) (20 μg/ml) for 16 h prior to challenge with VSV-Luc (MOI = 0.1) for 6 h. Luciferase assay was performed to monitor VSV replication levels. <b>(B)</b> Control siRNA- or EAP30 siRNA-transfected-Huh7-TLR3 cells were mock-treated or transfected with 2 μg of poly(I:C) for 16 h prior to HCV-JFH1 infection (MOI = 0.05) for 48 h. Intracellular HCV RNA levels (relative to 28S rRNA) were quantified by qPCR. <b>(C)</b> PH5CH8 cells transiently overexpressing control vector or EAP30 were incubated with poly(I:C) for 16 h prior to challenge with VSV-Luc (MOI = 0.1) for 6 h. Luciferase assay was performed to monitor VSV replication levels. <b>(D)</b> Control vector- or EAP30-overexpressing Huh7-TLR3 cells were mock-treated or transfected with poly(I:C) for 16 h prior to HCV-JFH1 infection (MOI = 0.05) for 48 h. Intracellular HCV RNA levels (relative to 28S rRNA) were quantified by qPCR.</p

The impairment in viral induction of type I and type III IFNs following EAP30 knockdown is attributed to an IRF3-dependent and NF-κB-independent mechanism.

<p><b>(A)</b> Activation of the IFN-β promoter, IRF3-dependent PRDIII-I element, and NF-κB-dependent PRDII element by SeV in PH5CH8 cells transfected with control (empty bars) or EAP30 (solid bars) siRNA. <b>(B)</b> Activity of the human IRF3 promoter (IRF3(-779)) in PH5CH8 cells transfected with control siRNA (empty bars) or EAP30 siRNA (solid bars), and mock-infected or infected with SeV. <b>(C)</b> qPCR analysis of IL-6, IL-8, and MIP-1β mRNA levels in PH5CH8 cells transfected with control siRNA or EAP30 siRNA, and mock-infected (empty bars)or infected with SeV (solid bars). <b>(D)</b> qPCR analysis of IFN-β, IFN-λ1, and IFN-λ2/3 expression in control siRNA- or EAP30 siRNA-transfected PH5CH8 cells, mock-infected (empty bars)or infected with SeV (solid bars). <b>(E)</b> Production of IFN antiviral activity in culture supernatants of PH5CH8 cells under experimental conditions of <b>(D)</b>. “*” and “**” denote statistical differences exist as compared with control siRNA-transfected cells with a <i>P</i>-value of < 0.05 and < 0.01, respectively.</p

EAP30 synergizes with IRF3 and CBP to induce IRF3-dependent antiviral gene expression, resulting in an enhanced antiviral response.

<p><b>(A)</b> HEK293 (5x10⁴) cells were transfected with 100 ng of each plasmid or various double/triple plasmid combinations as indicated, with control vector being added to keep the total amount (300 ng) of transfected DNA constant in each condition. 48 h later, cells were challenged with VSV-Luc (MOI = 0.1) for 6 h (except the mock group) followed by cell lysis and luciferase assay. <b>(B)</b> Immunoblot analysis of transfected EAP30, EAP20 (using anti-HA), CBP, IRF3 (using anti-FLAG) and endogenous actin under experimental conditions of (A) for each transfection groups. <b>(C-E)</b> qPCR analysis of IFN-β (C), OASL (D), and IFN-λ1 (E) mRNA levels in HEK293 cells transfected with different plasmid combinations similar to panel (A) for 48 h. “*”, “**”, and “***” denote statistical differences exist as compared with empty vector-transfected cells with a <i>P</i>-value of < 0.05, < 0.01, and <0.001, respectively.</p

Alternative sentencing and its comparison with other European Union countries

The main aim of my thesis is to develop an analysis of current legislation governing alternative sentences, their possibilities and limits comparing it with other European Union countries. The thesis also examines the proposals de lege ferenda that could be used as a benchmark and a guide for future amendments to the current regulation. The notion of alternative sentencing became a global trend in the last two to three decades; this is the result of, inter alia, of continuous increase in the number of inmates, prison overcrowding and congestion of the judicial apparatus. Crescent crime and new forms of crime (especially economic) hit the Czech Republic as a former socialist country in the post-revolutionary times, very assertive. The results of studies and language experts warn against lax approach in the form of increased storage imprisonment and criminal policy tightening. There is also talk about crisis imprisonment. It is clear that an unconditional sentence of imprisonment has its benefits, such as preventive effect, in my opinion, however, the left especially serious crime offenders and recidivist behavior: "Nesit summum malum dolor, malum certe est." Instead of intramural prison environment with significant social and deviant subculture find great potential in alternative prison sentences, which are..