Cigarette Smoke Suppresses Type I Interferon-Mediated Antiviral Immunity in Lung Fibroblast and Epithelial Cells

The objective of this study was to investigate the impact of cigarette smoke on innate antiviral defense mechanisms; specifically, we examined the effects of cigarette smoke on the induction of type I interferon (IFN). We observed a dose-dependent decrease in the ability of human lung fibroblast and epithelial cells to elicit an antiviral response against a viral double-strand RNA (dsRNA) mimic, polyI:C, in the presence of cigarette smoke-conditioned medium (SCM). Mechanistically, SCM decreases the expression of IFN-stimulated gene 15 (ISG15) and IFN regulatory factor-7 (IRF-7) transcripts and suppresses the nuclear translocation of key transcription factors, nuclear factor-κB (NF-κB) and IRF-3, after polyI:C stimulation. Furthermore, we provide evidence that the intercellular defense strategy against viral infection is also impaired. We observed a decrease in the ability of fibroblasts to elicit an antiviral state in response to IFN-β stimulation. This was associated with decreased nuclear translocation of phosphorylated Stat1 in response to IFN-β treatment. The effects elicited by SCM are reversible and are almost entirely abrogated in the presence of an antioxidant, such as glutathione. Our findings suggest that cigarette smoke affects the immediate-early, inductive, and amplification phases of the type I IFN response

Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist

The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-β promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response

HSV Infection Induces Production of ROS, which Potentiate Signaling from Pattern Recognition Receptors: Role for S-glutathionylation of TRAF3 and 6

The innate immune response constitutes the first line of defense against infections. Pattern recognition receptors recognize pathogen structures and trigger intracellular signaling pathways leading to cytokine and chemokine expression. Reactive oxygen species (ROS) are emerging as an important regulator of some of these pathways. ROS directly interact with signaling components or induce other post-translational modifications such as S-glutathionylation, thereby altering target function. Applying live microscopy, we have demonstrated that herpes simplex virus (HSV) infection induces early production of ROS that are required for the activation of NF-κB and IRF-3 pathways and the production of type I IFNs and ISGs. All the known receptors involved in the recognition of HSV were shown to be dependent on the cellular redox levels for successful signaling. In addition, we provide biochemical evidence suggesting S-glutathionylation of TRAF family proteins to be important. In particular, by performing mutational studies we show that S-glutathionylation of a conserved cysteine residue of TRAF3 and TRAF6 is important for ROS-dependent activation of innate immune pathways. In conclusion, these findings demonstrate that ROS are essential for effective activation of signaling pathways leading to a successful innate immune response against HSV infection

Hepatitis C Virus NS5A Targets the Nucleosome Assembly Protein NAP1L1 to Control the Interferon Response

Hepatitis C virus (HCV) is a hepatotropic virus affecting more than 150 million people worldwide. HCV establishes a chronic infection in the majority of cases, which leads to severe liver complications such as cirrhosis and hepatocellular carcinoma. Fortunately, recent potent direct acting antivirals can now cure the infection. However, such treatments can induce resistance, are extremely costly limiting their use to wealthier countries and are ineffective for the complications of the infection. Therefore, a better understanding of the interaction of HCV with the host cell remains a priority both to increase the armamentarium of antiviral drugs and to define the relationship between infection and malignant transformation. My study focuses on the mechanisms governing the evasion of the innate immune system, which are required to establish a chronic infection. The non-structural viral protein NS5A has the capacity to interact with a large number of cellular factors involved in promoting viral replication/assembly and in the cell antiviral response to HCV. Interaction of NS5A with the nucleosome assembly protein NAP1L1 has been recently characterized in my laboratory. NAP1L1 is a histone chaperone protein with various functions related to nuclear chromatin remodelling that impact on the regulation of cell cycle, on cell differentiation and on transcription. I have confirmed the interaction of NS5A with NAP1L1 in the cytoplasm and demonstrated the NS5A-dependent impairment of NAP1L1 nuclear translocation. Whole genome transcription analysis performed in NAP1L1 depleted hepatocytes indicated that its nuclear function might be essential for the transcriptional control of several interferon stimulated genes and the function of key innate immunity pathways. Indeed, I was able to demonstrate that NAP1L1 is a novel factor involved in the interferon response and specifically modulates TBK1/IKKε mediated IRF-3 phosphorylation and NF-κB levels. Hence, both the TLR3 and RIG-I/MDA5 pathways are affected by NAP1L1 depletion. I could further demonstrate that NAP1L1 controls the basal transcription of genes involved in the immune pathway and that it interacts with the adaptor protein MAVS, which is required for RIG-I/MDA5 signalling. In conclusion, by studying the interaction of the viral protein NS5A with the cellular factor NAP1L1 I could discover a novel mechanism of regulation of the innate response mediated by NAP1L1. These findings have wider implications for HCV and beyond, further highlighting the importance of studying viruses to uncover cellular functions

Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist

The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus-(SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response

Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist

The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-β promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response

Characterization of the non-structural (NSs) protein of tick-borne phleboviruses

In recent years, a number of newly discovered tick-borne viruses exhibiting a wide spectrum of diseases in humans have been ascribed to the Phlebovirus genus of the Bunyaviridae family. These viruses have a tripartite RNA genome composed of two negative-sense RNA segments (medium and large) and one ambisense segment (small), which encode four structural proteins and one non-structural protein (NSs). The NSs protein is the major virulence factor of bunyaviruses, and acts as an antagonist of a key component of the first line of defence against viral infections: the interferon (IFN) system (Bridgen et al., 2001; Weber et al., 2002). The work presented herein describes the characterization of tick-borne phlebovirus NSs proteins as IFN antagonists. The development of a reverse genetics system for the apathogenic tick-borne Uukuniemi phlebovirus (UUKV) enabled the recovery of infectious UUKV entirely from cDNA. A recombinant UUKV lacking NSs induced higher amounts of IFN in infected cells compared to wild-type UUKV, suggesting a role of NSs in modulating the IFN response. The weak IFN antagonistic activity of UUKV NSs was evident using transient transfection reporter assays in comparison to the NSs protein of either pathogenic Heartland virus (HRTV) or Severe fever with thrombocytopenia syndrome virus (SFTSV). The sensitivity of UUKV, HRTV and SFTSV to exogenous and virus-induced IFN, as well as their growth kinetics in IFN-competent cells were examined. The molecular mechanisms employed by UUKV, HRTV and SFTSV NSs proteins to evade antiviral immunity were investigated using reporter assays, immunofluorescence, and immunoprecipitation studies. Collectively, these assays showed that UUKV NSs was able to weakly inhibit IFN induction but not IFN signalling, through a novel interaction with MAVS (mitochondrial antiviral signalling protein). On the other hand, HRTV and SFTSV NSs proteins potently inhibited IFN induction through an interaction with TBK1, and type I but not type II IFN signalling via an interaction with STAT2. Finally, the development of a minigenome system for HRTV in conjunction with minigenomes developed for UUKV and SFTSV (Brennan et al., 2015) provided preliminary data to assess possible outcomes of tick-borne phlebovirus reassortment. In summary, the results described in this thesis offer insights into how tick-borne phlebovirus pathogenicity may be linked to the capacity of their NSs proteins to block the innate immune system. The data presented also illustrate the plethora of viral immune evasion strategies utilized by emerging phleboviruses, and provide an insight into the possibility of tick-borne phlebovirus reassortment

Redox-sensitive signaling following HSV recognition.

<p>(A) pDCs and (B) BMMs were treated with LNAC (3.2 mM) 30 min prior to infection with HSV-1 or 2 (MOI 3). Culture supernatants were harvested 16 h post infection, and levels of CCL5 were measured by ELISA. All data are shown as means of 3–5 replicates +/- st.dev. (C) MEFs were treated with LNAC (3.2 mM) 30 min prior to infection with HSV-1 or 2 (MOI 3). Total RNA was harvested after 6 h post-infection and IFN-β mRNA measured by real time PCR. Data is presented as means of triplicate measurements +/- st.dev. RU, relative units. (D, E) Mice were treated i.p. with LNAC (1.1 mmol/kg body weight) in saline or with saline alone followed 4 h later by infection with HSV-2 (5×10⁶ pfu). Eight and 24 hours post infection, serum and livers were harvested for measurement of type I IFN and viral load, respectively (n = 5). Data represents mean of 3–6 replicates +/- st.dev. (F, G) RAW264.7 macrophage-like cells were treated with LNAC (6.4 mM) for 30 min before infection with HSV-2 (3×10⁶ pfu/ml, MOI 3). (F) Total cell lysates were harvested at the indicated time points post infection, and phospho-IκBα was measured by Luminex. (G) The cells were fixed 3 h post infection, stained with anti-IRF-3 antibody and DAPI, and visualised by confocal microscopy. Cells were scored for nuclear IRF-3 staining. Data represent mean +/- st.dev. (H, I) Peritoneal macrophages from C57BL/6 WT and ASK1^-/- mice were cultured <i>in vitro</i> and infected with HSV-2 (3×10⁶ pfu/ml, MOI 3). Supernatants were harvested 16 h post infection, and the levels of CCL5 and IFN-α/β were measured. (J) C57BL/6 WT and ASK1^-/- mice were infected i.p. with 5×10⁶ pfu of HSV-2. Livers were isolated 3 days post infection and viral load in the organs was determined (n = 8). The data represent mean of multiple measurements +/- st.dev.</p