SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition

Abstract

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

Keywords: EMCV; HSV; MAVS; SIDT2; bystander immunity; double-stranded RNA; type I interferon; virus infection.

Figure 1. SIDT2 localises to late endosomes and interacts with internalised poly(I:C)

(A) MEFs stably expressing SIDT2-mCherry were transfected with markers for (i) early endosomes (EEA1-GFP), (ii) late endosomes (RAB7-GFP) and (iii) lysosomes (LAMP1-YFP), and imaged by confocal microscopy. Co-localization analysis of SIDT2-mCherry with endosomal markers was performed using FIJI software (see main text). Scale bar = 40 μm. (B) DC2.4 cells stably expressing SIDT2-mCherry were transfected with dsDNA-fluorescein or treated with poly(I:C)-fluorescein for 1 h and imaged by confocal microscopy. (C–D) Assessment of SIDT2-mCherry interactions with poly(I:C)-fluorescein and dsDNA-fluorescein in DC2.4 cells via FRET FLIM. ^** P < 0.01. Data is plotted as ± SEM and are representative of 3 independent experiments. See also Supplemental videos 1–4.

Figure 2. Loss of SIDT2 impairs innate immunity to EMCV infection

(A) Sidt2^+/+ and Sidt2^−/− mice (n=6–9) were infected with 50 PFU EMCV i.p. and survival monitored for 8 days. (B–C) Sidt2^+/+ and Sidt2^−/− mice (n=10–13) were sacrificed at 3 days p.i. and viral load was assessed in the heart and PECs by plaque assay. (D–E) Peritoneal lavage fluid and serum from Sidt2^+/+ and Sidt2^−/− mice (n=10–13) were collected at day 3 p.i. and IFN-β was measured via ELISA. (F–H) Serum IL-6, RANTES and IL-12 p40 from Sidt2^+/+ and Sidt2^−/− mice (n=10–13) at 3 days p.i. were measured using Bioplex bead assay. For panels F–H, data are plotted as mean ± SEM. ^* P < 0.05, ^*** P < 0.001, n.d = not detected.

Figure 3. Loss of SIDT2 impairs innate immunity to HSV-1 infection

(A) Vero cells were infected with 1 MOI GFP-tagged HSV-1 virus for 24 h, stained with J2 anti-dsRNA antibody (red) and DAPI (blue), and imaged by confocal microscopy. In the absence of HSV-1 infection, no dsRNA was evident (top panel), whereas following HSV-1 challenge dsRNA was readily observed in uninfected cells (bottom panel, arrows). Data are representative of three independent experiments. Scale bar = 20 μm. (B) Sidt2^+/+ and Sidt2^−/− MEFs were infected with 1 MOI mCherry-tagged HSV-1 virus for the indicated times and analysed by flow cytometry. Data is representative of 3 independent experiments. Error bars represent mean ± SEM. (C) Cell culture supernatant from Sidt2^+/+ and Sidt2^−/− MEFs infected with 1 MOI mCherry-tagged HSV-1 was collected at 96 h p.i. and IFN-β was measured via ELISA. (D) Sidt2^+/+ and Sidt2^−/− mice (n=13–15) were infected with 1×10⁷ PFU HSV-1 i.p. and survival monitored for 8 days. (E–F) Serum from Sidt2^+/+ and Sidt2^−/− mice (n=10–13) was collected at 8h and 16h p.i. respectively and serum IFNβ was measured via ELISA. (G–H) Serum IL-6 and RANTES from Sidt2^+/+ and Sidt2^−/− mice (n=10–13) at 16 h p.i. were measured using Bioplex bead assay. For panels E–H, data are plotted as mean ± SEM. ^* P < 0.05, ^*** P < 0.001, n.s. = not significant. Data represents the pooled results from 3 independent experiments. See also Figure S3.

Figure 4. Loss of SIDT2 impairs endosomal escape of internalised poly(I:C)

BMDCs from Sidt2^+/+ and Sidt2^−/− mice were treated with (A) poly(I:C)-rhodamine and (B) ³²P-labeled 500bp dsRNA for 60 min at either 4°C or 37°C, and internalisation assessed via flow cytometry or radioactivity measurement respectively. Results are representative of at least 2 independent experiments. For panel B, all treatments and measurements were made in triplicate, and data are plotted as mean ± SEM. (C–D) BMDCs from Sidt2^+/+ and Sidt2^−/− mice were treated with poly(I:C) for either 10 or 60 min, stained with J2 anti-dsRNA antibody (red) and DAPI (blue), and imaged by confocal microscopy. The proportion of each cell occupied by punctate dsRNA staining was quantified. (E–F) Sidt2^+/+ and Sidt2^−/− BMDCs were treated with poly(I:C) in association with the cationic polymer DEAE-dextran for 60 min. For panels C and E, images are representative of at least three independent experiments. For panels D and F, data are plotted as mean ± SEM and between 30–150 cells were assessed per time point. ^* P < 0.05. Scale bar = 10 μm. See also Figure S5.

Figure 5. Overexpression of SIDT2 enhances endosomal escape of poly(I:C)

(A) Doxycycline-inducible SIDT2-mCherry DC2.4 cells were treated with poly(I:C)-fluorescein for 1 h in the presence or absence of doxycycline and imaged by confocal microscopy. Scale bar = 10 μm (B) The proportion of each cell occupied by punctate poly(I:C)-fluorescein staining was individually quantified for >60 cells across multiple fields of view, and plotted as mean ± SEM. (C) Internalisation of poly(I:C)-fluorescein by SIDT2-mCherry⁺ and SIDT2-mCherry⁻ cells after 1 h at either 4°C or 37°C was assessed via flow cytometry. Data are representative of 3 independent experiments. ^****P < 0.0001. See also Figure S5.

Figure 6. SIDT2 is important for RLR- but not TLR-mediated IFN production in response to extracellular dsRNA

(A) Sidt2^+/+ and Sidt2^−/− mice were injected i.p with 50 μg poly(I:C) per 25 g body weight (n=7), and serum IFNβ measured at 3 h via ELISA. (B) Sidt2^+/+ and Sidt2^−/− mice (n=8) were injected i.p. with 300 μg poly(A:U) per 25 g body weight and serum IFNβ measured at 3 h via ELISA. (C) Sidt2^+/+ and Sidt2^−/− mice (n=8) were injected i.p. with 50 μg poly(I:C) per 25 g body weight and serum IFNλ measured at 3 h via ELISA (n=8). Data are plotted as mean ± SEM. (D) BMDMs from Sidt2^+/+ and Sidt2^−/− were stimulated with 10 μg/ml poly(I:C) for the indicated times and pIRF3^Ser386 and total IRF3 was assessed via immunoblotting. (E) Sidt2^+/+ and Sidt2^−/− MEFs were transfected with MAVS-YFP, treated with poly(I:C) for 1 h, and MAVS aggregation assessed via confocal microscopy. Scale bar = 40 μm. (F) Individual cells (>150 per condition) were scored for the appearance of MAVS aggregates. Data is representative of 3 independent experiments and error bars are plotted as mean ± SEM. ^* P < 0.05, n.s. = not significant, n.d = not detected.

Figure 7. SIDT2 is required for RLR-induced MAVS activation in bystander cells during HSV-1 infection

(A) PECs from Sidt2^+/+ and Sidt2^−/− mice infected with 1×10⁷ PFU at 16 h p.i. were stained with J2 dsRNA antibody (red) and DAPI (blue) and imaged by confocal microscopy. Scale bar = 10 μm. (B) The proportion of each cell occupied by punctate dsRNA staining was quantified using FIJI software. (C) Sidt2^+/+ and Sidt2^−/− MEFs stably expressing MAVS-YFP were infected with 1 MOI HSV-1-mCherry for 48 h, and imaged via confocal microscopy to assess for MAVS aggregation (arrows) in uninfected, bystander cells. Scale bar = 80 μm. (D) Individual cells were segmented and HSV-1-mCherry infected cells were excluded using FIJI software. Uninfected bystander cells (250 cells per condition) were scored for the appearance of MAVS aggregates. (E) Sidt2^+/+ (Cas9 only), Sidt2^−/− (Cas9 only), MAVS^−/− and Sidt2^−/− MAVS^−/− MEFs were infected with 1 MOI mCherry-tagged HSV-1, and IFNβ was measured in cell culture supernatant at 96 h p.i. via ELISA. (F) IL-6 and (G) TNF-α were measured in cell culture supernatant from shTLR3 and Sidt2^−/− shTLR3 MEFs infected with 1 MOI mCherry-tagged HSV-1 96h p.i. Sidt2^+/+ and Sidt2^−/− MEFs transduced with retroviral vector lacking shRNA were used as controls. IFN-β, IL-6 and TNF-α in non-infected cells were below the limit of assay detection. Data is representative of results from 2–3 independent experiments and expressed as the mean ± SEM of triplicate wells. ^* P < 0.05, ^** P < 0.01, n.s. = not significant. See also Figure S6.