RTN3 inhibits RIG-I-mediated antiviral responses by impairing TRIM25-mediated K63-linked polyubiquitination
- Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-Sen University, China
- Key Laboratory of Tropical Disease Control (Sun Yat-Sen University), Ministry of Education, China
- Zhongshan School of Medicine, Sun Yat-Sen University, China
Figures
Figure 1 with 1 supplement
RTN3 is upregulated and self-aggregates upon RNA viral infection.
(A) Immunoblot analysis of HEK293T cells infected with VSV-eGFP (MOI = 1) or treated with poly(I:C) (5 μg/ml) at the indicated timepoints. (B–C) mRNA levels of RTN3 and TNF in the same samples shown …
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Figure 1—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, D, and F.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data1-v2.zip
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Figure 1—source data 2
Excel file of RT-PCR measurements and description of statistical tests for panel B.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data2-v2.xlsx
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Figure 1—source data 3
Excel file of RT-PCR measurements and description of statistical tests for panel C.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data3-v2.xlsx
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Figure 1—source data 4
Excel file of RT-PCR measurements and description of statistical tests for panel E.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data4-v2.xlsx
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Figure 1—source data 5
Excel file of RT-PCR measurements and description of statistical tests for panel F.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-data5-v2.xlsx
Figure 1—figure supplement 1
RTN3 is upregulated and self-aggregates upon RNA viral infection.
(A–B) Immunoblot analysis of A549 cells treated with poly(I:C) (5 µg/ml) (A) or infected with VSV-eGFP (MOI = 1) (B) at the indicated timepoints. (C) mRNA levels of RTN3 in the same samples shown in …
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Figure 1—figure supplement 1—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, B, D, F, and H.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-figsupp1-data1-v2.zip
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Figure 1—figure supplement 1—source data 2
Excel file of RT-PCR measurements and description of statistical tests for panel C.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-figsupp1-data2-v2.xlsx
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Figure 1—figure supplement 1—source data 3
Excel file of RT-PCR measurements and description of statistical tests for panel E.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-figsupp1-data3-v2.xlsx
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Figure 1—figure supplement 1—source data 4
Excel file of images quantification and description of statistical tests for panel G.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig1-figsupp1-data4-v2.xlsx
Figure 2 with 1 supplement
RTN3 overexpression suppresses antiviral immune responses.
(A) Luciferase assays of HEK293T cells transfected with an ISRE luciferase reporter (ISRE-Luc), an IFNB luciferase reporter (IFNβ-Luc) or an NF-κB luciferase reporter (NF-κB-Luc) together with an …
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Figure 2—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panel E.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data1-v2.zip
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Figure 2—source data 2
Excel file of luciferase reporter assay measurements and description of statistical tests for panel A.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data2-v2.xlsx
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Figure 2—source data 3
Excel file of luciferase reporter assay measurements and description of statistical tests for panel B.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data3-v2.xlsx
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Figure 2—source data 4
Excel file of RT-PCR measurements and description of statistical tests for panel C.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data4-v2.xlsx
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Figure 2—source data 5
Excel file of blots quantification and description of statistical tests for panel E.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data5-v2.xlsx
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Figure 2—source data 6
Excel file of RT-PCR measurements and description of statistical tests for panel F.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-data6-v2.xlsx
Figure 2—figure supplement 1
RTN3 suppresses RNA virus-induced antiviral immune and inflammatory responses.
(A) Luciferase activity of HEK293T cells transfected with ISRE-Luc together with HA-Ev or increasing amounts of HA-RTN3 (+, ++) and followed by transfection with MDA5 CARDs as an activator. (B) …
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Figure 2—figure supplement 1—source data 1
Excel file of luciferase reporter assay measurements and description of statistical tests for panel A.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data1-v2.xlsx
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Figure 2—figure supplement 1—source data 2
Excel file of luciferase reporter assay measurements and description of statistical tests for panel B.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data2-v2.xlsx
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Figure 2—figure supplement 1—source data 3
Excel file of luciferase reporter assay measurements and description of statistical tests for panel C.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data3-v2.xlsx
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Figure 2—figure supplement 1—source data 4
Excel file of luciferase reporter assay measurements and description of statistical tests for panel D.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data4-v2.xlsx
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Figure 2—figure supplement 1—source data 5
Excel file of MTS assay measurements and description of statistical tests for panel E.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data5-v2.xlsx
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Figure 2—figure supplement 1—source data 6
Excel file of RT-PCR measurements and description of statistical tests for panel F.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data6-v2.xlsx
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Figure 2—figure supplement 1—source data 7
Excel file of RT-PCR measurements and description of statistical tests for panel G.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data7-v2.xlsx
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Figure 2—figure supplement 1—source data 8
Excel file of RT-PCR measurements and description of statistical tests for panel H.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data8-v2.xlsx
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Figure 2—figure supplement 1—source data 9
Excel file of RT-PCR primers list for panel H.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig2-figsupp1-data9-v2.xlsx
Figure 3
RTN3 knockdown enhances antiviral immune responses.
(A) RT-PCR analysis (top) and immunoblotting analysis (bottom) of RTN3 mRNA levels in PBMCs infected with Ctrl or RTN3 shRNA encoding lentivirus (shCtrl, shRNA #1, shRNA #2) for 12 hr and cultured …
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Figure 3—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panels A and B.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data1-v2.zip
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Figure 3—source data 2
Excel file of RT-PCR measurements and description of statistical tests for panel A.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data2-v2.xlsx
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Figure 3—source data 3
Excel file of blots quantification and description of statistical tests for panel B.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data3-v2.xlsx
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Figure 3—source data 4
Excel file of RT-PCR measurements and description of statistical tests for panel C.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data4-v2.xlsx
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Figure 3—source data 5
Excel file of RT-PCR measurements and description of statistical tests for panel D.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig3-data5-v2.xlsx
Figure 4 with 1 supplement
RTN3 separately interacts with TRIM25 and RIG-I and inhibits the K63-linked polyubiquitination of RIG-I.
(A) Coimmunoprecipitation (CoIP) and immunoblot analyses of HEK293T cells transfected with a plasmid encoding TRIM25 (Flag-TRIM25) or a Flag-tagged empty vector (Flag-Ev) together with GFP-tagged …
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Figure 4—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, B, C, F, G, and H.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig4-data1-v2.zip
Figure 4—figure supplement 1
RTN3 has no influence on the protein levels of RIG-I and TRIM25 but affects their K63-polyubiquitination.
(A) CoIP and immunoblot analyses of HEK293T cells transfected with HA-Ev or HA-RTN3 together with GFP tagged TRIM25 (GFP-TRIM25) or GFP-Ev for the indicated groups for 24 hr and followed by …
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Figure 4—figure supplement 1—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, B, C, D, E, F, G, H, and I.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
RTN3-mediated inhibition of antiviral responses is TRIM25-dependent.
(A) Immunoblot analysis of control HEK293T cells (HEK293T-shCtrl) and TRIM25 knockdown HEK293T cells (HEK293T-shTRIM25) transfected with HA-Ev or HA-RTN3 and followed by treated with poly(I:C) (5 …
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Figure 5—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panels A, B, and C.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig5-data1-v2.zip
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Figure 5—source data 2
Excel file of blots quantification and description of statistical tests for panel A.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig5-data2-v2.xlsx
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Figure 5—source data 3
Excel file of luciferase reporter assay measurements and description of statistical tests for panel D.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig5-data3-v2.xlsx
Figure 5—figure supplement 1
RTN3 inhibitory activity requires its transmembrane domain 1.
(A) CoIP and immunoblot analyses of HEK293T cells transfected with HA-Ub-K63, HA-RTN3 and its truncated mutants as in (A) together with GFP-RIG-I or GFP-Ev for the indicated groups for 24 hr and …
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Figure 5—figure supplement 1—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panel A, Band C.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig5-figsupp1-data1-v2.zip
Figure 6 with 1 supplement
RTN3 overexpression suppresses antiviral immune responses in mice.
(A) mRNA levels of Rtn3 were detected by RT-PCR in liver cells from mice treated with PBS, poly(I:C) (400 µg/mouse) or VSV-eGFP (1×107 PFU/g/mouse) for 12 hr. (B) Immunoblot analysis of liver cell …
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Figure 6—source data 1
Uncropped blots with bands labeled AI file and raw unedited blots files for panels B and D.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig6-data1-v2.zip
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Figure 6—source data 2
Excel file of CBA Mouse Inflammation Kit measurements and description of statistical tests for panel E.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
Determination of hydrodynamic injection efficiency and flow cytometry analysis of lymphocytes.
(A–B) Flow cytometry analysis for grouping of liver cell suspension and PBMCs from the same samples shown in (Figure 5F and G). Black and red boxes indicate the analyzed populations. (C) Macrophage, …
Figure 7 with 1 supplement
Rtn3 overexpression promotes inflammation resolution in mice.
(A) Microcopy analysis of liver and lung tissue sections from the same mice as in (Figure 5C) with hematoxylin and eosin (H and E) staining, and HA-Rtn3 exotic expression was visualized by …
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Figure 7—source data 1
Excel file of inflammation scores and description of statistical tests for panel B.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig7-data1-v2.xlsx
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Figure 7—source data 2
Excel file of IHC scores and description of statistical tests for panel C.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig7-data2-v2.xlsx
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Figure 7—source data 3
Excel file of inflammation scores and description of statistical tests for panel D.
- https://cdn.elifesciences.org/articles/68958/elife-68958-fig7-data3-v2.xlsx
Figure 7—figure supplement 1
Viral infection in the liver increases upon RTN3 overexpression.
(A–B) The EGFP-positive percentage in the liver cell suspensions and the fluorescent intensity for the same samples shown in Figure 5F and G were analyzed by flow cytometry. In (B), the data are …
Figure 8
The working model of the inhibition of RIG-I mediated antiviral responses by RTN3 Upon RNA virus infection, the RIG-I signalosome is activated by TRIM25-mediated K63-linked polyubiquitination, and then triggers IRF3 and NF-κB activation and the expression of interferon, cytokines and inflammatory factors.
On the other hand, RTN3 is up-regulated by acute viral infection and interferon/inflammatory factors in an ER stress-dependent manner. RTN3 is subsequently aggregated on the endoplasmic reticulum, …
