Astrocytes evoke a robust IRF7-independent type I interferon response upon neurotropic viral infection.

BACKGROUND: Type I interferons (IFN-I) are fundamental in controlling viral infections but fatal interferonopathy is restricted in the immune-privileged central nervous system (CNS). In contrast to the well-established role of Interferon Regulatory Factor 7 (IRF7) in the regulation of IFN-I response in the periphery, little is known about the specific function in the CNS. METHODS: To investigate the role for IRF7 in antiviral response during neurotropic virus infection, mice deficient for IRF3 and IRF7 were infected systemically with Langat virus (LGTV). Viral burden and IFN-I response was analyzed in the periphery and the CNS by focus formation assay, RT-PCR, immunohistochemistry and in vivo imaging. Microglia and infiltration of CNS-infiltration of immune cells were characterized by flow cytometry. RESULTS: Here, we demonstrate that during infection with the neurotropic Langat virus (LGTV), an attenuated member of the tick-borne encephalitis virus (TBEV) subgroup, neurons do not rely on IRF7 for cell-intrinsic antiviral resistance and IFN-I induction. An increased viral replication in IRF7-deficient mice suggests an indirect antiviral mechanism. Astrocytes rely on IRF7 to establish a cell-autonomous antiviral response. Notably, the loss of IRF7 particularly in astrocytes resulted in a high IFN-I production. Sustained production of IFN-I in astrocytes is independent of an IRF7-mediated positive feedback loop. CONCLUSION: IFN-I induction in the CNS is profoundly regulated in a cell type-specific fashion.

Rapid SARS-CoV-2 Adaptation to Available Cellular Proteases.

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2+ human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.

Interferon regulatory factor-1 protects from fatal neurotropic infection with vesicular stomatitis virus by specific inhibition of viral replication in neurons.

The innate immune system protects cells against invading viral pathogens by the auto- and paracrine action of type I interferon (IFN). In addition, the interferon regulatory factor (IRF)-1 can induce alternative intrinsic antiviral responses. Although both, type I IFN and IRF-1 mediate their antiviral action by inducing overlapping subsets of IFN stimulated genes, the functional role of this alternative antiviral action of IRF-1 in context of viral infections in vivo remains unknown. Here, we report that IRF-1 is essential to counteract the neuropathology of vesicular stomatitis virus (VSV). IFN- and IRF-1-dependent antiviral responses act sequentially to create a layered antiviral protection program against VSV infections. Upon intranasal infection, VSV is cleared in the presence or absence of IRF-1 in peripheral organs, but IRF-1-/- mice continue to propagate the virus in the brain and succumb. Although rapid IFN induction leads to a decline in VSV titers early on, viral replication is re-enforced in the brains of IRF-1-/- mice. While IFN provides short-term protection, IRF-1 is induced with delayed kinetics and controls viral replication at later stages of infection. IRF-1 has no influence on viral entry but inhibits viral replication in neurons and viral spread through the CNS, which leads to fatal inflammatory responses in the CNS. These data support a temporal, non-redundant antiviral function of type I IFN and IRF-1, the latter playing a crucial role in late time points of VSV infection in the brain.

Hepatitis C virus replication in mouse cells is restricted by IFN-dependent and -independent mechanisms.

BACKGROUND & AIMS: Current treatment strategies for hepatitis C virus (HCV) infection include pegylated interferon (IFN)-alfa and ribavirin. Approximately 50% of patients control HCV infection after treatment, but the broad range of patients' outcomes and responses to treatment, among all genotypes, indicates a role for host factors. Although the IFN system is important in limiting HCV replication, the virus has evolved mechanisms to circumvent the IFN response. However, direct, IFN-independent antiviral processes also might help control HCV replication. We examined the role of IFN-independent responses against HCV replication. METHODS: We analyzed replication of the subgenomic JFH1 replicon in embryonic fibroblasts and primary hepatocytes from mice with disruptions in genes encoding factors in the IFN-dependent and alternative antiviral pathways (signal transducers and activators of transcription 1 [STAT1], protein kinase R, interferon regulatory factors (IRF) IRF-1, IRF-3, IRF-5, IRF-7, mitochondrial antiviral signaling molecule [MAVS], and IFN receptor [IFNAR]). We also assessed the effects of expression of these factors by mouse primary hepatocytes on HCV replication. RESULTS: In addition to IRF-3- and IFN-mediated antiviral responses, IFN-independent, but IRF-1- and IRF-5-dependent mechanisms, restrict HCV replication in mouse embryonic fibroblasts. In primary hepatocytes these IFN-independent require MAVS and IRF-1. CONCLUSIONS: HCV replication is limited by interferon-mediated pathways as well pathways that are independent of type I IFNs. IRF1 and IRF5 control IFN-independent signaling events that lead to antiviral responses. We observed antiviral roles of IRF1 and IRF5 that were IFN-independent and cell-type specific. These mechanisms are important in controlling viruses that interfere with the IFN signaling because cells retain the ability to induce functional but local antiviral states through expression of interferon-stimulated genes.

IFN regulatory factor-1 bypasses IFN-mediated antiviral effects through viperin gene induction.

Viperin is an antiviral protein whose expression is highly upregulated during viral infections via IFN-dependent and/or IFN-independent pathways. We examined the molecular alterations induced by the transcriptional activator IFN regulatory factor (IRF)-1 and found viperin to be among the group of IRF-1 regulated genes. From these data, it was not possible to distinguish genes that are primary targets of IRF-1 and those that are targets of IRF-1-induced proteins, like IFN-beta. In this study, we show that IRF-1 directly binds to the murine viperin promoter to the two proximal IRF elements and thereby induces viperin expression. Infection studies with embryonal fibroblasts from different gene knock-out mice demonstrate that IRF-1 is essential, whereas the type I IFN system is dispensable for vesicular stomatitis virus induced viperin gene transcription. Further, IRF-1, but not IFN type I, mediates the induction of viperin transcription after IFN-gamma treatment. In contrast, IRF-1 is not required for IFN-independent viperin induction by Newcastle disease virus infection and by infection with a vesicular stomatitis virus mutant that is unable to block IFN expression and secretion. We conclude that the IRF-1 mediated type I IFN independent mechanism of enhanced viperin expression provides a redundant mechanism to protect cells from viral infections. This mechanism becomes important when viruses evade innate immunity by antagonizing the induction and function of the IFN system.

Tumor suppression by IFN regulatory factor-1 is mediated by transcriptional down-regulation of cyclin D1.

IFNs have been ascribed to mediate antitumor effects. IFN regulatory factor-1 (IRF-1) is a major target gene of IFNs. It inhibits cell proliferation and oncogenic transformation. Here, we show that 60% of all mRNAs deregulated by oncogenic transformation mediated by c-myc and H-ras are reverted to the expression levels of nontransformed cells by IRF-1. These include cell cycle-regulating genes. An indirect target is cyclin D1. Activation of IRF-1 decreased cyclin D1 expression and cyclin-dependent kinase 4 kinase activity concomitant with change in the levels of hyperphosphorylated retinoblastoma protein. These effects are mediated by inhibition of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway and a transcriptional repression of cyclin D1. As shown by in vitro assays and tumor growth in nude mice, IRF-1-mediated effects on cell cycle progression were found to be overridden by ectopic expression of cyclin D1. Conversely, decrease of cyclin D1 by RNA interference experiments prevents transformation and tumor growth. The data show that cyclin D1 is a key target for IRF-1-mediated tumor-suppressive effects.

ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNß. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNß-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.

IRF-1 expression is essential for natural killer cells to suppress metastasis.

IFN-γ promotes tumoral immune surveillance, but its involvement in controlling metastases is less clear. Using a mouse model of pulmonary metastases, we show that local IFN-γ treatment inhibits formation of metastases through its regulation of IRF-1 in tumor cells. IRF-1 is an IFN-γ-induced transcription factor pivotal in the regulation of infection and inflammation. IRF-1 blockade abolished the inhibitory effect of IFN-γ on tumor metastases, whereas ectopic expression of IRF-1 phenocopied the inhibitory effects of IFN-γ. IRF-1 did not affect the survival of tumor cells in the circulation or their infiltration into lungs, but it was essential to support the pulmonary attraction and activation of natural killer (NK) cells. Depleting NK cells from mice abolished the protective effect of IFN-γ or IRF-1 on metastases. In addition, cytotoxicity assays revealed that tumor cells expressing IRF-1 were targeted more effectively by NK cells than IRF-1 nonexpressing tumor cells. Moreover, NK cells isolated from lungs inoculated with IRF-1-expressing tumor cells exhibit a greater cytotoxic activity. Mechanistic investigations revealed that IRF-1-induced NK cell cytotoxicity was independent of perforin and granzyme B but dependent on the NK cell activating receptor DNAM-1. Taken together, our findings establish IRF-1 as an essential mediator of the cross-talk between tumor cells and NK cells that mediate immune surveillance in the metastatic niche.