A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes.

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-ß) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-ß-inducing virus. IFN-ß induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-ß induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.

HSPD1 interacts with IRF3 to facilitate interferon-beta induction.

The production of IFN- I (IFN-α/ß) is one of the earliest and most important host-protective responses. Interferon regulatory factor 3 (IRF3) is a critical transcriptional factor in the IFN-ß signaling pathway. Although significant progress has been achieved in the regulation of IRF3, the process may be more complicated than previously considered. In the present study, heat shock protein 60 (HSP60, HSPD1) was identified as a novel IRF3-interacting protein. Overexpression of HSPD1 facilitated the phosphorylation and dimerization of IRF3 and enhanced IFN-ß induction induced by SeV infection. In contrast, knockdown of endogenous HSPD1 significantly inhibited the signaling pathway. Furthermore, HSPD1 enhanced activation of the IFN-ß promoter mediated by RIG-I, MDA-5, MAVS, TBK1 and IKKε but not IRF3/5D, a mock phosphorylated form of IRF3. The present study indicated that HSPD1 interacted with IRF3 and it contributed to the induction of IFN-ß.

Applications of selected reaction monitoring (SRM)-mass spectrometry (MS) for quantitative measurement of signaling pathways.

Quantitative measurement of the major regulatory proteins in signaling networks poses several technical challenges, including low abundance, the presence of post-translational modifications (PTMs), and the lack of suitable affinity detection reagents. Using the innate immune response (IIR) as a model signaling pathway, we illustrate the approach of stable isotope dilution (SID)-selected reaction monitoring (SRM)-mass spectrometry (MS) assays for quantification of low abundance signaling proteins. A work flow for SID-SRM-MS assay development is established for proteins with experimentally observed MS spectra and for those without. Using the interferon response factor (IRF)-3 transcription factor as an example, we illustrate the steps in high responding signature peptide identification, SID-SRM-MS assay optimization, and evaluation. SRM assays for normalization of IIR abundance to invariant housekeeping proteins are presented. We provide an example of SID-SRM assay development for post-translational modification (PTM) detection using an activating phospho-Ser modified NF-κB/RelA transcription factor, and describe challenges inherent in PTM-SID-SRM-MS assay development. Application of highly qualified quantitative, SID-SRM-MS assays will enable a systems-level approach to understanding the dynamics and kinetics of signaling in host cells, such as the IIR.

Interferon regulatory factor-3 activation, hepatic interferon-stimulated gene expression, and immune cell infiltration in hepatitis C virus patients.

UNLABELLED: Interferon regulatory factor-3 (IRF-3) activation directs alpha/beta interferon production and interferon-stimulated gene (ISG) expression, which limits virus infection. Here, we examined the distribution of hepatitis C virus (HCV) nonstructural 3 protein, the status of IRF-3 activation, and expression of IRF-3 target genes and ISGs during asynchronous HCV infection in vitro and in liver biopsies from patients with chronic HCV infection, using confocal microscopy and functional genomics approaches. In general, asynchronous infection with HCV stimulated a low-frequency and transient IRF-3 activation within responsive cells in vitro that was associated with cell-to-cell virus spread. Similarly, a subset of HCV patients exhibited the nuclear, active form of IRF-3 in hepatocytes and an associated increase in IRF-3 target gene expression in hepatic tissue. Moreover, ISG expression profiles formed disease-specific clusters for HCV and control nonalcoholic fatty liver disease patients, with increased ISG expression among the HCV patients. We identified the presence of T cell and plasmacytoid dendritic cell infiltrates within all biopsy specimens, suggesting they could be a source of hepatic interferon in the setting of hepatitis C and chronic inflammatory condition. CONCLUSION: These results indicate that HCV can transiently trigger IRF-3 activation during virus spread and that in chronic HCV, IRF-3 activation within infected hepatocytes occurs but is limited.

Cell-cell fusion induced by measles virus amplifies the type I interferon response.

Measles virus (MeV) infection is characterized by the formation of multinuclear giant cells (MGC). We report that beta interferon (IFN-beta) production is amplified in vitro by the formation of virus-induced MGC derived from human epithelial cells or mature conventional dendritic cells. Both fusion and IFN-beta response amplification were inhibited in a dose-dependent way by a fusion-inhibitory peptide after MeV infection of epithelial cells. This effect was observed at both low and high multiplicities of infection. While in the absence of virus replication, the cell-cell fusion mediated by MeV H/F glycoproteins did not activate any IFN-alpha/beta production, an amplified IFN-beta response was observed when H/F-induced MGC were infected with a nonfusogenic recombinant chimerical virus. Time lapse microscopy studies revealed that MeV-infected MGC from epithelial cells have a highly dynamic behavior and an unexpected long life span. Following cell-cell fusion, both of the RIG-I and IFN-beta gene deficiencies were trans complemented to induce IFN-beta production. Production of IFN-beta and IFN-alpha was also observed in MeV-infected immature dendritic cells (iDC) and mature dendritic cells (mDC). In contrast to iDC, MeV infection of mDC induced MGC, which produced enhanced amounts of IFN-alpha/beta. The amplification of IFN-beta production was associated with a sustained nuclear localization of IFN regulatory factor 3 (IRF-3) in MeV-induced MGC derived from both epithelial cells and mDC, while the IRF-7 up-regulation was poorly sensitive to the fusion process. Therefore, MeV-induced cell-cell fusion amplifies IFN-alpha/beta production in infected cells, and this indicates that MGC contribute to the antiviral immune response.

Cytomegalovirus induces interferon-stimulated gene expression and is attenuated by interferon in the developing brain.

Cytomegalovirus (CMV) is considered the most common infectious agent causing permanent neurological dysfunction in the developing brain. We have previously shown that CMV infects developing brain cells more easily than it infects mature brain cells and that this preference is independent of the host B- and T-cell responses. In the present study, we examined the innate antiviral defenses against mouse (m) and human (h) CMVs in developing and mature brain and brain cells. mCMV infection induced interferon (IFN)-stimulated gene expression by 10- to 100-fold in both glia- and neuron-enriched cultures. Treatment of primary brain cultures with IFN-alpha, -beta, and -gamma or a synthetic RNA, poly(I:C), reduced the number of mCMV-infected cells, both in older cells and in fresh cultures from embryonic mouse brains. When a viral dose that killed almost all unprotected cells was used, IFN-protected cells had a natural appearance, and when they were tested with whole-cell patch clamp recording, they appeared physiologically normal with typical resting membrane potentials and action potentials. mCMV infection increased expression of representative IFN-stimulated genes (IFIT3, OAS, LMP2, TGTP, and USP18) in both neonatal and adult brains to similarly large degrees. The robust upregulation of gene expression in the neonatal brain was associated with a much higher degree of viral replication at this stage of development. In contrast to the case for downstream gene induction, CMV upregulated IFN-alpha/beta expression to a greater degree in the adult brain than in the neonatal brain. Similar to the case with cultured brain cells, IFN treatment of the developing brain in vivo depressed mCMV replication. In parallel work with cultured primary human brain cells, IFN and poly(I:C) treatment reduced hCMV infection and prevented virus-mediated cell death. These results suggest that coupling IFN administration with current treatments may reduce CMV infections in the developing brain.

SOCS1-negative feedback of STAT1 activation is a key pathway in the dsRNA-induced innate immune response of human keratinocytes.

Toll-like receptor (TLR)3 is a receptor for virus-associated double-stranded RNA, and triggers antiviral immune responses during viral infection. Epidermal keratinocytes express TLR3 and provide an innate immune defense against viral infection. Since the intracellular regulatory mechanism is unknown, we hypothesized that the signal transducers and activators of transcription (STAT)-suppressors of cytokine signaling (SOCS) system regulates the innate immune response of keratinocytes. Treatment with polyinosinic-polycytidylic acid (poly(I:C)) resulted in the rapid translocation of IFN regulatory factor (IRF)-3 into the nucleus, followed by phosphorylation of STAT1 and STAT3. The activation of STATs by poly(I:C) probably occurs in an indirect fashion, through poly(I:C)-induced IFN. We infected cells with the dominant-negative forms of STAT1 (STAT1F), STAT3 (STAT3F), and SOCS1 using adenovirus vectors. Infection with STAT1F suppressed the induction of macrophage inflammatory protein (MIP)-1alpha by poly(I:C), whereas STAT3F had a minimal effect, which indicates that STAT1 mediates MIP-1alpha induction. SOCS1, which is a negative feedback regulator of STAT1 signaling, was induced by treatment with poly(I:C). SOCS1 infection inhibited the phosphorylation of STAT1 and significantly reduced poly(I:C)-induced MIP-1alpha production. Furthermore, STAT1-SOCS1 regulated poly(I:C)-induced TLR3 and IRF-7 expression. However, SOCS1 did not affect NF-kappaB signaling. Thus, the STAT1-SOCS1 pathway regulates the innate immune response via TLR3 signaling in epidermal keratinocytes.

Fusion protein between protein ZZ and red fluorescent protein DsRed and its application to immunoassays.

In the present study, a red fluorescent protein (DsRed) from the coral Discosoma was fused to the C-terminus of protein ZZ, a synthetic artificial IgG-Fc-fragment-binding protein derived from the B-domain of staphylococcal Protein A. The chimaeric protein, tagged with six histidine residues at the N-terminus, was expressed in Escherichia coli and easily purified by one-step Ni2+-chelating affinity chromatography. Its fluorescence and IgG-binding activities were validated using fluorescence-spectrum analysis, ELISA and dot-blot analysis. Furthermore, in subsequent dot-blotting immunoanalysis of glutathione S-transferase and tumour necrosis factor-alpha, and immunofluorescent microscopy assay of interferon regulatory factor 3, the chimaeric protein enabled effective detection of target molecules. Compared with fluorescence-conjugated antibodies, ZZ-DsRed is less susceptible to photobleaching and easy to produce. In addition, unlike HRP (horseradish peroxidase)-conjugated antibodies, using ZZ-DsRed needs no addition of a chromogenic reagent. Our results indicate that ZZ-DsRed shows a wide and promising application potential in immunological detection as a substitute for fluorescent or HRP-conjugated anti-IgGs.