Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity
- Beijing Institute of Basic Medical Sciences, China
- Anhui Medical University, China
- Institute of Immunology, Medical School of Henan University, China
- Department of Oncology, First Hospital of Jilin University, China
- Beijing Institute of Brain Disorders, Laboratory of Brain Disorders, Ministry of Science and Technology, Collaborative Innovation Center for Brain Disorders, Capital Medical University, China
Figures
Figure 1 with 1 supplement
Tim-3 inhibits VSV replication in macrophages.
(A) Peritoneal macrophages were isolated from wild-type (WT) mice and infected with vesicular stomatitis virus (VSV) for the indicated hours. Then the Tim-3 mRNA expression was examined by …
-
Figure 1—source code 1
Flox gene amplication.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig1-code1-v2.zip
-
Figure 1—source code 2
Cre gene amplication.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig1-code2-v2.zip
-
Figure 1—source code 3
Genotype identification.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig1-code3-v2.zip
-
Figure 1—source code 4
Phenotype identification.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig1-code4-v2.zip
-
Figure 1—source data 1
Tim-3 mRNA expression in macrophages in response to VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig1-data1-v2.zip
-
Figure 1—source data 2
Tim-3 protein expression in macrophage in response to VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig1-data2-v2.zip
-
Figure 1—source data 3
VSV induces Tim-3 phosphorylation .
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig1-data3-v2.zip
-
Figure 1—source data 4
Tim-3 signaling promotes VSV replication in macrophage .
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig1-data4-v2.zip
Figure 1—figure supplement 1
Development and identification of Tim-3 knockout mice.
(A) Mating protocol. (B and C) Mice tails were cut and DNA was extracted. Flox gene (B) and Cre gene (C) segments were PCR amplified and determined by electrophoresis. (D) Genotype examination to …
Figure 2 with 1 supplement
Tim-3 interacts with and inhibits NF90.
(A and B) Protein complex of Tim-3 and NF90 overexpressed in cells. HEK293T cells were transfected with plasmids encoding HA-Tim-3, Flag-NF90, and Myc-Tim-3 for 24 hr, immunoprecipitated with HA or …
-
Figure 2—source code 1
Tim-3-WT were transfected into HEK293T cells.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig2-code1-v2.zip
-
Figure 2—source code 2
Candidates interacts with Tim-3.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig2-code2-v2.zip
-
Figure 2—source data 1
Tim-3 interacts with NF90.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig2-data1-v2.zip
-
Figure 2—source data 2
NF90 interacts with Tim-3.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig2-data2-v2.zip
-
Figure 2—source data 3
Tim-3 interacts with NF90 via intracellular tail.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig2-data3-v2.zip
-
Figure 2—source data 4
Tim-3 inhibits NF90.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig2-data4-v2.zip
Figure 2—figure supplement 1
MS data showing Tim-3-interacting proteins in macrophages.
(A and B) Co-immunoprecipitation and mass spectrometry for screening of proteins interacting with Tim-3. Tim-3-WT were transfected into HEK293T cells for 24 hr, cell lysates were immunoprecipitated …
Figure 3 with 1 supplement
Tim-3 promotes the ubiquitination of NF90 at the DZF domain.
(A) Tim-3 enhances the ubiquitination of NF90 in macrophages in response to vesicular stomatitis virus (VSV) challenges. Peritoneal macrophages in wild-type (WT) and Tim-3-TG mice were infected with …
-
Figure 3—source code 1
Tim-3 mediates NF90 ubiquitination.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig3-code1-v2.zip
-
Figure 3—source data 1
Tim-3 promotes ubiquitination of NF90.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig3-data1-v2.zip
-
Figure 3—source data 2
Tim-3 promotes k48-linked ubiquitination of NF90.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig3-data2-v2.zip
-
Figure 3—source data 3
NF90 canbe ubiquitinated at the DZF domain.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig3-data3-v2.zip
-
Figure 3—source data 4
Tim-3 promotes NF90 ubiquitination at the DZF domain.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig3-data4-v2.zip
Figure 3—figure supplement 1
Peritoneal macrophages from WT and Tim-3-TG mice were infected with VSV for 4 hr and treated with MG132 (20 ug/ml) for 6 hr.
The cells were lysed, immunoprecipitated with NF90 (ILF3) antibody, and detected by western blot using the indicated antibodies.
Figure 4 with 1 supplement
Involvement of TRIM47 in Tim-3-mediated NF90 degradation.
(A) E3 ligases identified by mass spectrometry for top peptide hits (defined by Mascot score) associated with NF90 ubiquitination. (B) TRIM47 promotes NF90 degradation in a proteasome-dependent …
-
Figure 4—source code 1
Silence of TRIM47 in macrophages.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig4-code1-v2.zip
-
Figure 4—source code 2
TRIM47 promotes NF90 degradation.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig4-code2-v2.zip
-
Figure 4—source data 1
TRIM47 promotes NF90 degradation.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig4-data1-v2.zip
-
Figure 4—source data 2
Tim-3 synthesizes with TRIM47 to promote NF90 degradation.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig4-data2-v2.zip
-
Figure 4—source data 3
Tim-3 promotes TRIM47 expression.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig4-data3-v2.zip
-
Figure 4—source data 4
Tim-3 promotes TRIM47 expression.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig4-data4-v2.zip
-
Figure 4—source data 5
Tim-3promotes k63-linked ubiquitination of TRIM47.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig4-data5-v2.zip
Figure 4—figure supplement 1
TRIM47 knockdown increases expression of NF90.
(A) Quantitative reverse transcription PCR (qPCR) analysis of TRIM47 mRNA expression in RAW264.7 macrophages 48 hr after transfection with TRIM47 siRNA. (B) Western blot analysis of the protein …
Figure 5 with 1 supplement
Tim-3 recruits TRIM47 to DZF domain of NF90 within which Lys297 is a critical site for TRIM47-mediated K48-linked ubiquitination and degradation of NF90.
(A and B) The intracellular domain of Tim-3 and the DZF domain of NF90 interact with TRIM47 respectively. HEK293T cells were transfected with the indicated plasmids for 24 hr and treated with MG132 …
-
Figure 5—source code 1
Lys297 is a critical site for ubiquitination of NF9.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig5-code1-v2.zip
-
Figure 5—source data 1
Tim-3 interacts with TRIM47 via its intracellular tail.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig5-data1-v2.zip
-
Figure 5—source data 2
NF90 interacts with TRIM47 via its DZF domain.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig5-data2-v2.zip
-
Figure 5—source data 3
Tim-3 promotes TRIM47 mediated ubiquitination of NF90.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig5-data3-v2.zip
-
Figure 5—source data 4
Lys297 is a critical site for TRIM47-mediated ubiquitination of NF90.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig5-data4-v2.zip
-
Figure 5—source data 5
Lys297 is a critical site for TRIM47-mediated ubiquitination of NF90-DZF.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig5-data5-v2.zip
Figure 5—figure supplement 1
Lys297 of NF90 is a critical site in TRIM47-mediated K48-linked ubiquitination and degradation of NF90.
Co-immunoprecipitation analysis of the polyubiquitination of NF90-DZF (WT) and its mutants in HEK293T cells transfected with plasmids encoding HA–ubiquitin (K48). Data are representative of three …
Figure 6 with 1 supplement
Tim-3 selectively inhibits the phosphorylation of PKR and eIF2a and decreases the expression of SG markers G3BP1 and TIA-1 in macrophages.
(A) Lysates from wild-type (WT) and Tim-3 gene (Havcr2) knockout (Tim-3KO) peritoneal macrophages infected with vesicular stomatitis virus (VSV) were analyzed by immunoblotting for the indicated …
-
Figure 6—source code 1
TRIM47 knock down increases TIA-1,G3BP1 expression and decrease VSV expression.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig6-code1-v2.zip
-
Figure 6—source data 1
Tim-3 selectively inhibits the phosphorylation of PKR and eIF2a.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig6-data1-v2.zip
-
Figure 6—source data 2
Tim-3 increases the expression of SGs markers G3BP1 and TIA-1 in macrophages.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig6-data2-v2.zip
Figure 6—figure supplement 1
TRIM47 knockdown enhances antiviral immunity in macrophages.
TRIM47 was silenced in RAW264.7 macrophages as in Figure 2—figure supplement 1. (A and B) Quantitative reverse transcription PCR (qPCR) analysis of TIA-1 and G3BP1 expression in RAW264.7 macrophages …
Figure 7
Tim-3 deficiency upregulates G3BP1 and TIA-1 and protects mice from VSV infection.
(A, B, D, E, G, and H) Detection of mRNA transcription of G3BP1 and TIA-1 in organs and peritoneal macrophages by quantitative reverse transcription PCR (qPCR) after wild-type (WT) and Tim-3KO mice …
-
Figure 7—source data 1
Tim-3 knowdown increases G3BP1expression in spleen with VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data1-v2.zip
-
Figure 7—source data 2
Tim-3 knowdown increases TIA-1 expression in spleen with VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data2-v2.zip
-
Figure 7—source data 3
Tim-3 knowdown decreases VSV expression in spleen with infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data3-v2.zip
-
Figure 7—source data 4
Tim-3 knowdown increases G3BP1 expression in lung with VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data4-v2.zip
-
Figure 7—source data 5
Tim-3 knowdown increases TIA-1 expression in lung with VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data5-v2.zip
-
Figure 7—source data 6
Tim-3 knowdown decreases VSV expression in lung with VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data6-v2.zip
-
Figure 7—source data 7
Tim-3 knowdown increases G3BP1 expression in peritoneal macrophages with VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data7-v2.zip
-
Figure 7—source data 8
Tim-3 knowdown increases TIA-1 expression in peritoneal macrophages with VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data8-v2.zip
-
Figure 7—source data 9
Tim-3 knowdown decreases VSV expression in peritoneal macrophages with VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data9-v2.zip
-
Figure 7—source data 10
Tim-3 deficiency protects mice from VSV infection.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data10-v2.zip
-
Figure 7—source data 11
Tim-3 knowdown attenuated tissue damage.
- https://cdn.elifesciences.org/articles/66501/elife-66501-fig7-data11-v2.zip
Figure 8
Schematic diagram of how Tim-3 inhibits NF90-SG pathway in macrophages during infection.
Upon vesicular stomatitis virus (VSV) infection, Tim-3 is activated and upregulated. The activation of Tim-3 in turn recruited the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and …
Additional files
-
Supplementary file 1
Sequences of the primers used for PCR.
- https://cdn.elifesciences.org/articles/66501/elife-66501-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/66501/elife-66501-transrepform-v2.pdf
