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The active form of TBK1 localizes to the Golgi apparatus after RLR stimulation. (A) HeLa cells were either left unstimulated or infected with Sendai virus (SeV) for 6 or 8 h. Cells were then fractionated as described in Additional file 1A, and samples were analyzed by immunoblotting with antibodies against the indicated proteins. EEA1, kinectin, LAMP2, GAPDH, syntaxin-6, and VDAC served as loading and purity controls for endosomes, the endoplasmic reticulum, lysosomes, the cytosol, the Golgi apparatus, and mitochondria, respectively. (Ub)n, polyubiquitin. * Indicates non-specific bands. (B–E) HeLa cells were either left unstimulated (control) or infected with SeV for 6 h (+ SeV). The indicated proteins were then analyzed by immunofluorescence staining. The Golgi apparatus was labeled with an antibody against GM130, whereas the mitochondria were identified by immunostaining for cytochrome c. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. (F) HeLa cells were either left unstimulated or infected with SeV for 6 or 8 h. Cell lysates (Lys.) were then subjected to immunoprecipitation (IP) with an antibody against TBK1. Samples were then analyzed by immunoblotting with antibodies against the indicated proteins. * Indicates non-specific bands. (PDF 2378 kb)

OPTN silencing impairs IRF3 signaling after RLR or TLR3 activation. (A) HEK293T cells were transfected with a control nonspecific siRNA (NS) or with five individual OPTNspecific siRNAs (OPTN A, B, C, D and E) or a NEMO-specific siRNA. The cells were then transfected, 48 h later, with an IFNβ promoter reporter or with a NF-κB reporter and the Renilla luciferase gene as an internal control. Then, 24 h after transfection, cells were either left unstimulated (Unstim) or infected with Sendai virus (+SeV) for 7 h. Luciferase assays were performed and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with NS siRNA-transfected cells in Student’s t-test). RLU, relative luminescence units. ns, not significant. (B) HEK293T cells were transfected with a control nonspecific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN A and OPTN B) or a NEMO-specific siRNA. Then, 72 h later, cells were either left unstimulated or infected with Sendai virus (SeV) for 6 or 8 h. Cell lysates were analyzed by immunoblotting with antibodies against the indicated proteins. * indicates nonspecific bands. (C) As in (B) but with HeLa cells. (D) HEK293T cells were transfected with a control nonspecific siRNA (NS) or with two individual OPTN-specific siRNAs (OPTN A and OPTN B) or a NEMO-specific siRNA. Cells were either left unstimulated (Unstim) or infected with VSV (+VSV) for 7 h. Luciferase assays were performed and the results were normalized against Renilla luciferase activity. (E) As in (B) but HEK293T cells were infected with VSV. (F) HEK293T-TLR3 cells were transfected as in (A). Cells were either left unstimulated (Unstim) or stimulated with poly(I:C) (10 μg/ml) for 7 h. Luciferase assays were then performed. (PDF 703 kb)

Transfection of MEFs with poly(I:C) triggers TBK1 accumulation at the centrosome at late time points. (A) MEFs were either left untreated (MOCK) or transfected with LMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). TBK1 staining was then analyzed by immunofluorescence. The Golgi apparatus was labeled with an antibody against GM130, whereas TBK1 was detected with a rabbit monoclonal antibody. Scale bars, 10 μm. (B–E) MEFs were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 μg/mL) for 4 h (trPoly(I:C)). The indicated proteins were analyzed by immunofluorescence staining with specific antibodies. The Golgi apparatus was labeled with an antibody against GM130, whereas centrosomes were labeled with an antibody against pericentrin. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. (PDF 1624 kb)

Influenza virus RNA promoter panhandle structures are believed to be sensed by RIG-I. The occurrence of mismatches in this dsRNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-I in vivo, affecting RNA/RIG-I complex formation, and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing.

p-TBK1S172 localizes to the Golgi apparatus then migrates to the centrosome after RLR activation. (A) Scheme of the subcellular fractionation procedure for obtaining the various membrane fractions in Figs. 1a, 2a, and Additional file 2A. (B) MEFs were either left unstimulated (control) or infected with Sendai virus (SeV) for 6 h (+ SeV). p-TBK1S172 staining was then analyzed by immunofluorescence. The Golgi apparatus was identified by labeling with an antibody raised against giantin. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. (C) Isolated Golgi membranes from uninfected or SeV-infected MEFs were analyzed by immunoblotting with antibodies against the indicated proteins. ° Corresponds to the p-TBK1 detection from a previous immunoblot. (D) MEFs were either left unstimulated (control) or infected with SeV for 6 or 9 h (+ SeV). p-TBK1S172 staining was then analyzed by immunofluorescence. The Golgi apparatus was identified by labeling with an antibody raised against GM130. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. (E) MEFs were either left unstimulated (control) or infected with SeV for 9 h (+ SeV). p-TBK1S172 staining was then analyzed by immunofluorescence. The centrosomes were stained with an antibody raised against pericentrin. Scale bars, 10 μm. On the right, enlargement of the framed zone in the overlay. (PDF 759 kb)

The order Mononegavirales includes three virus families that replicate in the cytoplasm: the Paramyxoviridae, composed of two subfamilies, the Paramyxovirinae and Pneumovirinae, the Rhabdoviridae and the Filoviridae. These viruses, also called non-segmented negative-strand RNA viruses (NNV), contain five to ten tandemly linked genes, which are separated by conserved junctional sequences that act as mRNA start and poly(A)/stop sites. For the NNV, downstream mRNA synthesis depends on termination of the upstream mRNA, and all NNV RNA-dependent RNA polymerases reiteratively copy ("stutter" on) a short run of template uridylates during transcription to polyadenylate and terminate their mRNAs. The RNA-dependent RNA polymerase of a subset of the NNV, all members of the Paramyxovirinae, also stutter in a very controlled fashion to edit their phosphoprotein gene mRNA, and Ebola virus, a filovirus, carries out a related process on its glycoprotein mRNA. Remarkably, all viruses that edit their phosphoprotein mRNA are also governed by the "rule of six", i.e. their genomes must be of polyhexameric length (6n+0) to replicate efficiently. Why these two seemingly unrelated processes are so tightly linked in the Paramyxovirinae has been an enigma. This paper will review what is presently known about these two processes that are unique to viruses of this subfamily, and will discuss whether this enigmatic linkage could be due to the phenomenon of RNA virus error catastrophe.