This study highlights IL-26 as a new player in the inflammatory and antiviral immune responses associated with chronic HCV infection.

The inhibitor of jB kinase e (IKKe) is pivotal for an efficient innate immune response to viral infections and has been recognized as breast cancer oncogene. The antiviral function of IKKe involves activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-jB, thus inducing the expression of type I IFN. Here, we have identified two novel splice variants of human IKKe, designated IKKe-sv1 and IKKe-sv2, respectively. Interestingly, RT-PCR revealed quantitatively different isoform expression in PBMC from different individuals. Moreover, we found cell type-and stimulus-specific protein expression of the various splice variants. Overexpression of full-length wt IKKe (IKKe-wt) leads to the activation of NF-jB-as well as IRF3-driven luciferase reporter genes. Although none of the splice variants activates IRF3, IKKe-sv1 still activates NF-jB, whereas IKKe-sv2 is also defective in NF-jB activation. Both splice variants form dimers with IKKe-wt and inhibit IKKe-wt-induced IRF3 signaling including the antiviral activity in a dominant-negative manner. The lack of IRF3 activation is likely caused by the failure of the splice variants to interact with the adapter proteins TANK, NAP1, and/or SINTBAD. Taken together, our data suggest alternative splicing as a novel regulatory mechanism suitable to shift the balance between different functions of IKKe.Key words: IFN . Innate immunity . NF-kB pathway . Protein/protein interactions . Signal transductionSupporting Information available online IntroductionViral infections are recognized by the innate immune system, which is essential for the subsequent initiation of adaptive immunity. Invading viruses are sensed by pattern-recognition receptors (PRR) recognizing pathogen-associated molecular patterns such as singleor double-stranded RNA. These PRR comprise TLR with endosomal/lysosomal localization like TLR3 and cytoplasmic receptors such as the retinoic acid-inducible protein I and melanoma differentiation-associated gene 5. Activation of these PRR engages intracellular signaling cascades leading to the secretion of type I IFN, which are important anti-viral cytokines ultimately facilitating viral clearance [1,2]. The signal transduction pathways leading to type I IFN expression involve activation of the serine/threonine kinases TANK-binding kinase 1 (TBK-1), also known as NF-kB activating kinase NAK [3], and inhibitor of kB kinase e (IKKe), also known as IKKi [4]. After virus infection, both kinases are activated and phosphorylate the transcription factor IFN regulatory factor 3 (IRF3), leading to homo-dimerization, translocation into the nucleus, and activation of promoters containing the IRF3 binding site termed IFN-stimulated response element [5,6]. For IRF3 activation after triggering of different PRR, the three related scaffold proteins NAP1, TANK, and SINTBAD are essential [7][8][9], whereas the use of a distinct scaffold protein depends on the respective stimulus activating the TBK1/IKKe pathway [10]. Ultimately, the formation of a multisubunit complex c...

Interleukin-26 (IL-26) belongs to the IL-10 cytokine family, is produced by activated T cells, and targets epithelial target cells for signal transduction. Here, we describe the IL-26 effects on the infection of culture cells with recombinant vesicular stomatitis virus (VSV), human cytomegalovirus (HCMV), and herpes simplex virus type 1 (HSV-1) expressing green fluorescent protein. After pre-incubation with recombinant IL-26 and at low multiplicity of infection, VSV showed strongly enhanced infection and replication rates as measured for infectivity, for transcript levels, and for protein expression. Control proteins did not affect VSV infection. The IL-26 effect was independent of the IL-26 receptor and neutralized by anti-IL-26 serum. Pre-incubation of VSV was much more efficient than pre-incubation of the target cells to enhance virus infection. IL-26 increased virus adsorption to target cells as shown by quantitative reverse-transcription PCR. In contrast, the infection of IL-26-treated human fibroblasts with HCMV was inhibited and the infection by HSV-1 was not altered by IL-26. Thus, IL-26 differentially modulates the infection by different enveloped viruses.

Interleukin-26 (IL-26) is a member of the IL-10 cytokine family due to sequence homology. IL-26 was discovered, since the gene is strongly overexpressed in T cells which are growth transformed by herpesvirus saimiri. The IL-26 gene maps to human chromosome 12q15 between the genes for two other T-cellular class-II cytokines, namely interferon-γ (lFN-γ) and lL-22. IL-26, IL-22, and IFN-γ are co expressed by activated T cells and, especially, by Th17 cells. IL-26 forms homodimers and adheres to glycosaminoglycans on cell surfaces, presumably due to its positive charge. IL-26 specifically targets the lL-26-specific heterodimeric receptor complex consisting of IL-20R1 and IL-10R2 which is typically expressed on epithelial cells such as colon carcinoma cells or keratinocytes. IL-26 stimulation induces STAT1 and STAT3 phosphorylation, CD54 surface expression, and cytokine secretion as shown for IL-8 and IL-10. IL-26 seems to act as a cell surface-associated and rather proinflammatory T-cell cytokine at the epithelial barrier, possibly linking T-cell response with epithelial functions.

In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (⌬aa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results. H erpes zoster is caused by endogenous varicella-zoster virus (VZV) reactivation, especially in the elderly or in patients with immunodeficiencies. The disease occurs in 5 to 32% of transplant patients (1) and is related to significant morbidity and mortality, with fatality rates of up to 28% (1-3). The efficacy of antiviral therapy was demonstrated by multiple randomized controlled studies. Acyclovir (ACV), its prodrug valacyclovir, famciclovir (the prodrug of penciclovir [PCV]), and (E)-5-(2-bromovinyl)-2=-deoxyuridine (brivudine [BVDU]) are approved as therapeutics in many countries to treat VZV (4). These nucleoside analogues are phosphorylated by the viral thymidine kinase (TK) (open reading frame 36 [ORF36]) and cellular kinases to form a triphosphate that blocks viral DNA polymerase (Pol) (ORF28) by acting as competitive inhibitors and/or DNA chain terminators. Acyclovir resistance has been reported in immunocompromised, but not in immunocompetent, patients (5). If a patient shows therapeutic failure within 7 to 10 days, drug susceptibility should be evaluated (4, 6, 7), and alternative drugs are required. Foscarnet (FOS) and cidofovir (CDV), both inhibitors of Pol (8, 9), act independently of the viral TK and are recomm...