Summary RIG-I is a cytosolic pathogen recognition receptor that initiates immune responses against RNA viruses. Upon viral RNA recognition, anti-viral signalling requires RIG-I redistribution from the cytosol to membranes where it binds the adaptor protein, MAVS. Here we identify the mitochondrial targeting chaperone protein, 14-3-3ε, as a RIG-I-binding partner and essential component of a translocation complex or “translocon” containing RIG-I, 14-3-3ε, and the TRIM25 ubiquitin ligase. The RIG-I translocon directs RIG-I redistribution from the cytosol to membranes where it mediates MAVS-dependent innate immune signalling during acute RNA virus infection. 14-3-3ε is essential for the stable interaction of RIG-I with TRIM25, which facilitates RIG-I ubiquitination and initiation of innate immunity against hepatitis C virus and other pathogenic RNA viruses. Our results define 14-3-3ε as a key component of a RIG-I translocon required for innate antiviral immunity.

The severe acute respiratory syndrome coronavirus accessory protein ORF6 antagonizes interferon signaling by blocking karyopherin-mediated nuclear import processes. Viral nuclear import antagonists, expressed by several highly pathogenic RNA viruses, likely mediate pleiotropic effects on host gene expression, presumably interfering with transcription factors, cytokines, hormones, and/or signaling cascades that occur in response to infection. By bioinformatic and systems biology approaches, we evaluated the impact of nuclear import antagonism on host expression networks by using human lung epithelial cells infected with either wild-type virus or a mutant that does not express ORF6 protein. Microarray analysis revealed significant changes in differential gene expression, with approximately twice as many upregulated genes in the mutant virus samples by 48 h postinfection, despite identical viral titers. Our data demonstrated that ORF6 protein expression attenuates the activity of numerous karyopherin-dependent host transcription factors (VDR, CREB1, SMAD4, p53, EpasI, and Oct3/4) that are critical for establishing antiviral responses and regulating key host responses during virus infection. Results were confirmed by proteomic and chromatin immunoprecipitation assay analyses and in parallel microarray studies using infected primary human airway epithelial cell cultures. The data strongly support the hypothesis that viral antagonists of nuclear import actively manipulate host responses in specific hierarchical patterns, contributing to the viral pathogenic potential in vivo. Importantly, these studies and modeling approaches not only provide templates for evaluating virus antagonism of nuclear import processes but also can reveal candidate cellular genes and pathways that may significantly influence disease outcomes following severe acute respiratory syndrome coronavirus infection in vivo.

Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4′ phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo-and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0-and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4′ phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0-and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach-ACP and suggest how the protein changes to accommodate its myriad binding partners.Acyl carrier proteins participate in a wide variety of biosynthetic processes, including the synthesis of fatty acids (1), toxins (2), oligosaccharides (3,4), polyketides (5,6), biotin (7), and depsipeptides (8). They ferry small molecules between enzymes involved in biosynthesis by covalent linkage as a thioester to the thiol group of 4′-phosphopantetheine ( Figure 1). This prosthetic group is attached by a post-translational modification to a serine (S38 in spinach ACP 1 ) in the middle a conserved Asp-Ser-Leu (DSL) sequence. Whereas the sequences of different acyl carrier proteins are divergent, they share a similar four-helix bundle topology and often can be interchanged with full activity in reactions in vitro. This prompts the question: are ACPs simply passive molecules that constantly present their cargo to enzymes, or do protein-protein and protein-ligand interactions affect the utility of ACP as an enzyme substrate?One well-studied biosynthetic process involving ACP is the desaturation of the fatty acid stearate by the enzyme stearoyl-ACP Δ 9 -desaturase (Δ9D). This enzyme, in a reaction dependent on O 2 and reducing-equivalent, acts on ACP with an attached stearate (18:0-ACP) † This work was supported by the National Institutes of Health Grants R01 GM-50853 to B.G.F. and R01 GM-58667 to J.L.M. NMR data were collected at the National Magnetic Resonance Facility at Madison (NMRFAM), which is supported by grants from the NIH Center for Research Resources Biomedical Research Technology Program (P41 RR-02301) and the National Institutes of General Medical Sciences (P41 GM-GM66326) with additional instrumentation purchased with funds from the University of Wisconsin, the NSF Biological Instrumentation Program, the NIH Shared Instrumentat...

Several respiratory viruses, including influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV), produce more severe disease in the elderly, yet the molecular mechanisms governing age-related susceptibility remain poorly studied. Advanced age was significantly associated with increased SARS-related deaths, primarily due to the onset of early-and late-stage acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. Infection of aged, but not young, mice with recombinant viruses bearing spike glycoproteins derived from early human or palm civet isolates resulted in death accompanied by pathological changes associated with ARDS. In aged mice, a greater number of differentially expressed genes were observed than in young mice, whose responses were significantly delayed. Differences between lethal and nonlethal virus phenotypes in aged mice could be attributed to differences in host response kinetics rather than virus kinetics. SARS-CoV infection induced a range of interferon, cytokine, and pulmonary wound-healing genes, as well as several genes associated with the onset of ARDS. Mice that died also showed unique transcriptional profiles of immune response, apoptosis, cell cycle control, and stress. Cytokines associated with ARDS were significantly upregulated in animals experiencing lung pathology and lethal disease, while the same animals experienced downregulation of the ACE2 receptor. These data suggest that the magnitude and kinetics of a disproportionately strong host innate immune response contributed to severe respiratory stress and lethality. Although the molecular mechanisms governing ARDS pathophysiology remain unknown in aged animals, these studies reveal a strategy for dissecting the genetic pathways by which SARS-CoV infection induces changes in the host response, leading to death.

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