Abstract
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 IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-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.
Highlights
Interferons (IFNs) are divided into three multigene families
The antibody’s variable regions of heavy (VH) and VL sequences were fused with a linker, generating a single-chain variable fragment
Triple staining revealed a lattice structure typical for the endoplasmic reticulum (ER). These results indicate that the intrabody-antigen complex was localized inside the ER
Summary
Interferons (IFNs) are divided into three multigene families (type I, II and III). The type I interferon family comprises the highest number of members: IFNαs, IFNβ, IFNε, IFNƬ, IFNκ, IFNω, IFNδ and IFNξ respectively [1]. Type I IFNs play a major role in the immune response during acute viral and bacterial infections and take part in induction of tumor cell death and inhibition of angiogenesis [2,3,4]. The type I family members IFNα and IFNβ are produced by almost all cells after contact with microbial products Their synthesis is induced after binding of danger signals (PAMPs or DAMPs) to some PRRs, especially TLR 7, 9 and RIG-I-like receptors [4]. Binding of type I IFNs to their receptor (IFNAR) induces multiple downstream signalling pathways leading to activation of a large number of IFN-stimulated genes (ISGs) in infected and neighbouring cells [2, 4]. Genes are induced that encode cytokines and chemokines, antibacterial effectors and pro-apoptotic and anti-apoptotic molecules [8]
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