Figure 1.
STING sequences through vertebrate evolution.
A. Multiple alignment of STING sequences from zebrafish and other vertebrates. Identical positions are boxed in black, conservative positions in grey and block of similar residues in light grey. The residues of the 5 putative transmembrane (TM) regions and the putative RXR ER retention motifs found in zebrafish STING sequence are boxed. Zebrafish (HE856619, this study), Danio rerio; human (NP_938023), Homo sapiens; mouse (NP_082537), Mus musculus; chicken (E1C7U0), Gallus gallus; xenopus (NP_001106445), Xenopus tropicalis. B. NJ phylogenetic tree of vertebrate STING. The tree was based on multiple alignments of full-length and partial STING amino-acid sequences from fish and other vertebrates. The tree is drawn to scale. Full-length sequence accession numbers are the following: EPC (HE856620, this study), Pimephales promelas; goldfish (JF970229), Carassius auratus, the others are listed above and partial STING amino-acid sequences were deduced from the following EST sequences: salmon (GE786872), Salmo salar; weather loach (BJ827384), Misgurnus anguillicandatus. C. Conserved synteny around the TMEM173 gene in zebrafish, mouse and human. The location of the different markers and the chromosomes involved are indicated for the different species.
Figure 2.
Zebrafish STING is a strong antiviral protein.
EPC cells were transfected with 2 µg of pcDNA-STING, and pcDNA-MAVS or an empty vector (pcDNA) as positive and negative controls, respectively. At 48 h posttransfection, EPC cells were infected with a recombinant rVHSV-Tom expressing the tdTomato fluorescent protein at an MOI of 1 and incubated at 15°C. Cell monolayers were visualized under a UV-visible light microscope at 24 h postinfection (A) and then stained with crystal violet 4 days postinfection (B). The culture supernatants from cells infected with rVHSV-Tom were collected at 0, 24 and 96 h postinfection and the viral titer was determined by plaque assay on EPC cells (C). Each time point was represented by three independent experiments, and each virus titration was done in duplicate. Means are shown. The standard errors were calculated and the error bars are shown. Asterisks indicate significant difference (*p<0.01 and **p<0.001) as determined by Student’s t test.
Figure 3.
Overexpression of STING induces an antiviral immunity against a DNA virus.
EPC cells were transfected with 2 µg of pcDNA-STING, and pcDNA-MAVS or an empty vector (pcDNA) as positive and negative controls, respectively. At 48 h posttransfection, EPC cells were infected with a DNA virus of the Iridoviridae family, i.e., EHNV, at MOI of 1, 0.1 and 0.01 (A and C). The culture supernatants were collected at 0, 24, 48 and 72 h postinfection and the viral titer was determined by plaque assay on EPC cells (B and D). Cell monolayers were then stained with crystal violet either at 24 h postinfection (A) or 4 days postinfection (C) depending to the MOI used, as indicated. Each time point was represented by three independent experiments, and each virus titration was done in duplicate. Means are shown together with the standard errors. Asterisks indicate significant difference (*p<0.01 and **p<0.001) as determined by Student’s t test.
Figure 4.
Overexpression of STING induced IFN and ISG expression.
EPC cells were transfected with 2 µg of pcDNA vector encoding STING, or MAVS as a positive control, or an empty vector (pcDNA) as a negative control. At 48 h posttransfection, EPC were infected or not with VHSV at an MOI of 1 and incubated at 15°C for 24 h before total RNA extraction. Quantitative Real-time RT-PCR was conducted using primers targeting IFN (A), RIG-I (B) and Viperin (C). The β-actin gene was used as an internal control to normalize the cDNA template and to do real-time PCR calculations. SD of triplicate experiments has been calculated. Asterisks indicate significant difference (*p<0.05, **p<0.01 and ***p<0.001) as determined by Student’s t test.
Figure 5.
STING antiviral response is mediated by IRF3.
EPC cells were co-transfected with 2 µg of pcDNA-IRF3-Cter encoding a dominant-negative mutant of IRF3, and 2 µg of pcDNA-STING, pcDNA-MAVS or an empty vector. At 48 h posttransfection, EPC cells were infected with a recombinant rVHSV-Tom expressing the tdTomato fluorescent protein at an MOI of 1 and incubated at 15°C. The culture supernatants were collected at 0, 24 and 96 h postinfection and the viral titer was determined by plaque assay on EPC cells. Cell monolayers were then stained with crystal violet either at 96 h postinfection. Each time point was represented by three independent experiments, and each virus titration was done in duplicate. Means are shown together with the standard errors. “ns” indicate non-significant difference (p>0.05) as determined by Student’s t test.
Figure 6.
STING and MAVS mediate RIG-I induction of interferon.
EPC cells were transfected with 1 µg of pIFNproLUC reporter in combination with various plasmid constructs (1 µg each, except for dominant-negative mutants of MAVS and STING combinations where 0.5 µg of each plasmid was used) as mentioned under each histogram. An empty vector (pcDNA) was added in some experiments to keep the total amount of transfected DNA constant (3 µg total DNA for 5×106 cells). In the condition were pRIG-I Nter-eGFP was not present in the transfection mixture, a peGFP vector was added. At 24 h and 48 h posttransfection, eGFP and luciferase signals were determined. Values of luciferase activities were normalized to the levels of eGFP fluorescence. The fold induction was calculated as the ratio of stimulated versus unstimulated (pcDNA alone) samples. Means of three independent experiments are shown together with the standard errors. Student’s t-test was used for statistical analysis, and asterisk indicate significant differences (*p<0.05 and **p<0.01).
Figure 7.
Localization of zebrafish STING to endoplasmic reticulum.
peGFP-STING or peGFP-MAVS and peGFP, as negative controls, were transfected together with a plasmid encoding the Red Fluorescent protein fused to a reticulum endoplasmic location signal (RFP-KDEL) into EPC cells (A). The mitochondria were in vivo stained with a red MitoTracker (B). The cells were imaged by microscopy 24 h post-transfection. The yellow staining in the overlay image indicates colocalization of STING and RFP-KDEL (A) or MAVS and MitoTracker (B).
Figure 8.
STING and MAVS are in close vicinity in mitochondrial-ER contact regions.
EPC cells were cotransfected with 1 µg of peGFP-STING and 1 µg of pCherry-MAVS. At 48 h posttransfection, EPC were infected with VHSV at an MOI of 1 and incubated at 15°C for 24 h before imaging by confocal microscopy. For both panels the main images show a section of the cell monolayer in the xy plane at the z position indicated by the grey arrow head in the xz plane (small top panel) and the yz plane (small right side panel). Orthogonal projections of confocal sections shown in the top and right side panels are in the cutting plane indicated by the white and the blue lines corresponding to the xz and yz planes, respectively. Zoomed insets of boxed areas in merged images are also presented.