Stephane Daffis

2′- O methylation of the viral mRNA cap evades host restriction by IFIT family members

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2′-O positions of the 5′ guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2′-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2′-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2′-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2′-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2′-O methylation of the 5′ cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2′-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

Toll-Like Receptor 3 Has a Protective Role against West Nile Virus Infection

ABSTRACT Protection against West Nile virus (WNV) infection requires rapid viral sensing and the generation of an interferon (IFN) response. Mice lacking IFN regulatory factor 3 (IRF-3) show increased vulnerability to WNV infection with enhanced viral replication and blunted IFN-stimulated gene (ISG) responses. IRF-3 functions downstream of several viral sensors, including Toll-like receptor 3 (TLR3), RIG-I, and MDA5. Cell culture studies suggest that host recognizes WNV in part, through the cytoplasmic helicase RIG-I and to a lesser extent, MDA5, both of which activate ISG expression through IRF-3. However, the role of TLR3 in vivo in recognizing viral RNA and activating antiviral defense pathways has remained controversial. We show here that an absence of TLR3 enhances WNV mortality in mice and increases viral burden in the brain. Compared to congenic wild-type controls, TLR3−/− mice showed relatively modest changes in peripheral viral loads. Consistent with this, little difference in multistep viral growth kinetics or IFN-α/β induction was observed between wild-type and TLR3−/− fibroblasts, macrophages, and dendritic cells. In contrast, a deficiency of TLR3 was associated with enhanced viral replication in primary cortical neuron cultures and greater WNV infection in central nervous system neurons after intracranial inoculation. Taken together, our data suggest that TLR3 serves a protective role against WNV in part, by restricting replication in neurons.

IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1−/− mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1−/− mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection.

Cell-Specific IRF-3 Responses Protect against West Nile Virus Infection by Interferon- Dependent and -Independent Mechanisms

Interferon regulatory factor (IRF)-3 is a master transcription factor that activates host antiviral defense programs. Although cell culture studies suggest that IRF-3 promotes antiviral control by inducing interferon (IFN)-β, near normal levels of IFN-α and IFN-β were observed in IRF-3−/− mice after infection by several RNA and DNA viruses. Thus, the specific mechanisms by which IRF-3 modulates viral infection remain controversial. Some of this disparity could reflect direct IRF-3-dependent antiviral responses in specific cell types to control infection. To address this and determine how IRF-3 coordinates an antiviral response, we infected IRF-3−/− mice and two primary cells relevant for West Nile virus (WNV) pathogenesis, macrophages and cortical neurons. IRF-3−/− mice were uniformly vulnerable to infection and developed elevated WNV burdens in peripheral and central nervous system tissues, though peripheral IFN responses were largely normal. Whereas wild-type macrophages basally expressed key host defense molecules, including RIG-I, MDA5, ISG54, and ISG56, and restricted WNV infection, IRF-3−/− macrophages lacked basal expression of these host defense genes and supported increased WNV infection and IFN-α and IFN-β production. In contrast, wild-type cortical neurons were highly permissive to WNV and did not basally express RIG-I, MDA5, ISG54, and ISG56. IRF-3−/− neurons lacked induction of host defense genes and had blunted IFN-α and IFN-β production, yet exhibited only modestly increased viral titers. Collectively, our data suggest that cell-specific IRF-3 responses protect against WNV infection through both IFN-dependent and -independent programs.

Interferon Regulatory Factor IRF-7 Induces the Antiviral Alpha Interferon Response and Protects against Lethal West Nile Virus Infection

ABSTRACT Type I interferon (IFN-α/β) comprises a family of immunomodulatory cytokines that are critical for controlling viral infections. In cell culture, many RNA viruses trigger IFN responses through the binding of RNA recognition molecules (RIG-I, MDA5, and TLR-3) and induction of interferon regulatory factor IRF-3-dependent gene transcription. Recent studies with West Nile virus (WNV) have shown that type I IFN is essential for restricting infection and that a deficiency of IRF-3 results in enhanced lethality. However, IRF-3 was not required for optimal systemic IFN production in vivo or in vitro in macrophages. To begin to define the transcriptional factors that regulate type I IFN after WNV infection, we evaluated IFN induction and virus control in IRF-7−/− mice. Compared to congenic wild-type mice, IRF-7−/− mice showed increased lethality after WNV infection and developed early and elevated WNV burdens in both peripheral and central nervous system tissues. As a correlate, a deficiency of IRF-7 blunted the systemic type I IFN response in mice. Consistent with this, IFN-α gene expression and protein production were reduced and viral titers were increased in IRF-7−/− primary macrophages, fibroblasts, dendritic cells, and cortical neurons. In contrast, in these cells the IFN-β response remained largely intact. Our data suggest that the early protective IFN-α response against WNV occurs through an IRF-7-dependent transcriptional signal.

Induction of IFN-β and the Innate Antiviral Response in Myeloid Cells Occurs through an IPS-1-Dependent Signal That Does Not Require IRF-3 and IRF-7

Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-α/β) induction and innate immune defenses after virus infection. Prior studies in mice with single deletions of the IRF-3 or IRF-7 genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed reduced IFN-α levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we generated IRF-3−/−× IRF-7−/− double knockout (DKO) mice, analyzed WNV pathogenesis, IFN responses, and signaling of innate defenses. Compared to wild type mice, the DKO mice exhibited a blunted but not abrogated systemic IFN response and sustained uncontrolled WNV replication leading to rapid mortality. Ex vivo analysis showed complete ablation of the IFN-α response in DKO fibroblasts, macrophages, dendritic cells, and cortical neurons and a substantial decrease of the IFN-β response in DKO fibroblasts and cortical neurons. In contrast, the IFN-β response was minimally diminished in DKO macrophages and dendritic cells. However, pharmacological inhibition of NF-κB and ATF-2/c-Jun, the two other known components of the IFN-β enhanceosome, strongly reduced IFN-β gene transcription in the DKO dendritic cells. Finally, a genetic deficiency of IPS-1, an adaptor involved in RIG-I- and MDA5-mediated antiviral signaling, completely abolished the IFN-β response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-β gene regulation after WNV infection in myeloid cells is IPS-1-dependent but does not require full occupancy of the IFN-β enhanceosome by canonical constituent transcriptional factors.

The Innate Immune Adaptor Molecule MyD88 Restricts West Nile Virus Replication and Spread in Neurons of the Central Nervous System

ABSTRACT Type I interferons (IFN-α/β) control viral infection by triggering the expression of genes that restrict transcription, translation, replication, and assembly. Many viruses induce IFN responses after recognition by cytoplasmic or endosomal RNA sensors (RIG-I-like RNA helicases [RLR] and Toll-like receptors [TLR]), which signal through the cognate adaptor signaling molecules IPS-1, TRIF, and MyD88. Recent studies have demonstrated that IPS-1-dependent induction of IFN-α/β downstream of RLR recognition restricts West Nile virus (WNV) infection in many cell types, whereas TRIF-dependent TLR3 signaling limits WNV replication in neurons. Here, we examined the contribution of MyD88 signaling to the control of WNV by evaluating IFN induction and virus replication in genetically deficient cells and mice. MyD88−/− mice showed increased lethality after WNV infection and elevated viral burden primarily in the brain, even though little effect on the systemic type I IFN response was observed. Intracranial inoculation studies corroborated these findings, as WNV spread more rapidly in the central nervous system of MyD88−/− mice, and this phenotype preceded the recruitment of inflammatory leukocytes. In vitro, increased WNV replication was observed in MyD88−/− macrophages and subsets of neurons but not in myeloid dendritic cells. MyD88 had an independent effect on recruitment of monocyte-derived macrophages and T cells into the brain that was associated with blunted induction of the chemokines that attract leukocytes. Our experiments suggest that MyD88 restricts WNV by inhibiting replication in subsets of cells and modulating expression of chemokines that regulate immune cell migration into the central nervous system.

A temporal role Of Type I interferon signaling in CD8+ T Cell maturation during acute West Nile virus infection

A genetic absence of the common IFN- α/β signaling receptor (IFNAR) in mice is associated with enhanced viral replication and altered adaptive immune responses. However, analysis of IFNAR-/- mice is limited for studying the functions of type I IFN at discrete stages of viral infection. To define the temporal functions of type I IFN signaling in the context of infection by West Nile virus (WNV), we treated mice with MAR1-5A3, a neutralizing, non cell-depleting anti-IFNAR antibody. Inhibition of type I IFN signaling at or before day 2 after infection was associated with markedly enhanced viral burden, whereas treatment at day 4 had substantially less effect on WNV dissemination. While antibody treatment prior to infection resulted in massive expansion of virus-specific CD8+ T cells, blockade of type I IFN signaling starting at day 4 induced dysfunctional CD8+ T cells with depressed cytokine responses and expression of phenotypic markers suggesting exhaustion. Thus, only the later maturation phase of anti-WNV CD8+ T cell development requires type I IFN signaling. WNV infection experiments in BATF3 -/- mice, which lack CD8-α dendritic cells and have impaired priming due to inefficient antigen cross-presentation, revealed a similar effect of blocking IFN signaling on CD8+ T cell maturation. Collectively, our results suggest that cell non-autonomous type I IFN signaling shapes maturation of antiviral CD8+ T cell response at a stage distinct from the initial priming event.

The role of myristoylation in the membrane association of the Lassa virus matrix protein Z.

The Z protein is the matrix protein of arenaviruses and has been identified as the main driving force for budding. Both LCMV and Lassa virus Z proteins bud from cells in the absence of other viral proteins as enveloped virus-like particles. Z accumulates near the inner surface of the plasma membrane where budding takes place. Furthermore, biochemical data have shown that Z is strongly membrane associated. The primary sequence of Z lacks a typical transmembrane domain and until now it is not understood by which mechanism Z is able to interact with cellular membranes. In this report, we analyzed the role of N-terminal myristoylation for the membrane binding of Lassa virus Z. We show that disruption of the N-terminal myristoylation signal by substituting the N-terminal glycine with alanine (Z-G2A mutant) resulted in a significant reduction of Z protein association with cellular membranes. Furthermore, removal of the myristoylation site resulted in a relocalization of Z from a punctuate distribution to a more diffuse cellular distribution pattern. Finally, treatment of Lassa virus-infected cells with various myristoylation inhibitors drastically reduced efficient Lassa virus replication. Our data indicate that myristoylation of Z is critical for its binding ability to lipid membranes and thus, for effective virus budding.

The Naturally Attenuated Kunjin Strain of West Nile Virus Shows Enhanced Sensitivity to the Host Type I Interferon Response

ABSTRACT The host determinants that contribute to attenuation of the naturally occurring nonpathogenic strain of West Nile virus (WNV), the Kunjin strain (WNVKUN), remain unknown. Here, we show that compared to a highly pathogenic North American strain, WNVKUN exhibited an enhanced sensitivity to the antiviral effects of type I interferon. Our studies establish that the virulence of WNVKUN can be restored in cells and mice deficient in specific interferon regulatory factors (IRFs) or the common type I interferon receptor. Thus, WNVKUN is attenuated primarily through its enhanced restriction by type I interferon- and IRF-3-dependent mechanisms.