Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells
- Cancer Research UK, London Research Institute, United Kingdom
- Utrecht University, Netherlands
Figures
Figure 1 with 2 supplements
IFN-α/β induction requires EMCV replication.
(A) EMCV and IAV RNA genomes were extracted from purified viral particles and tested at the indicated doses in an IFN-β promoter luciferase reporter assay in HEK293 cells in the presence of …
Figure 1—figure supplement 1
LGP2 and MDA5 are required for IFN-α/β production in response to EMCV.
(A and B) MEFs of the indicated genotypes were either not infected (NI), infected with EMCV or IAV at an MOI of 1, or transfected with 1 μg of in vitro transcribed RNA (IVT RNA). Ifit1 (A) and Ifnb1 …
Figure 1—figure supplement 2
Ribavirin abrogates EMCV RNA infectivity but does not decrease IFN-β reporter activity in 293 cells.
(A) HEK 293 cells were either left untreated or treated with 100 units/ml of IFN for 16 hr. RNA was then extracted and the expression of MDA5, LGP2, and RIG-I were analysed by RT-PCR and normalised …
Figure 2
LGP2 pulldown captures MDA5 agonistic RNA from EMCV-infected cells.
(A) Schematic representation of the experimental setup for LGP2 immunoprecipitation (IP). Precipitation efficiency was routinely verified by immunoblotting with an anti-FLAG antibody; an example is …
Figure 3 with 2 supplements
The L antisense RNA region is enriched in LGP2 pulldowns from EMCV-infected cells.
(A) RNA from LGP2 IP, control (ctrl) IP, or total RNA (input) from EMCV-infected cells (Figure 2A,B) was sequenced. Reads corresponding to human or EMCV sequences are shown as a percentage of the …
Figure 3—figure supplement 1
Comparison of read-distribution along the EMCV genome for LGP2-associated RNA and input material.
Strand-specific analysis of sequencing results from LGP2 IP vs input RNA from EMCV-infected cells (Figure 3). Number of reads per million obtained from Illumina sequencing were mapped to their …
Figure 3—figure supplement 2
LGP2 directly binds L region antisense RNA.
(A) Schematic representation of the experimental setup. Total RNA purified from HeLa cells infected with EMCV (input) was mixed with recombinant FLAG-tagged LGP2 before immunoprecipitation with an …
Figure 4
In vitro transcribed L AS RNA triggers an MDA5-dependent IFN response.
(A–C) L antisense (AS) (A), L sense (B) or IVT RNA (C) sequences were in vitro transcribed and all RNA except from the control IVT RNA (C) were CIP treated to remove any 5′ phosphates. The indicated …
Figure 5 with 1 supplement
The L region of EMCV is required for the generation of LGP2-associated stimulatory RNA.
(A) Schematic representation of the L region of EMCV genome and L region mutant viruses used in this study. The crosses indicate the position of the two point mutations in EMCV ZnC19AC22A. The …
Figure 5—figure supplement 1
200 ng of RNA isolated from HeLa cells infected with EMCV WT, ZnC19AC22A or ΔL at MOI 1 for 16 hr (HeLa EMCV RNA) was transfected into MDA5-sufficient or MDA5-deficient bone marrow-derived DCs in presence of ribavirin.
Transfection with ribavirin only and IVT RNA was used as negative and positive controls, respectively. Supernatants were harvested 16 hr later and mIFN-α levels measured by ELISA. ND = non detected. …
Figure 6
L region RNA is required for IFN-α/β production in response to EMCV.
(A–C) IFNAR1-deficient GM-CSF bone marrow-derived DCs were either not infected (NI) or infected with the indicated viruses at an MOI of 1 or 10. Levels of Ifit1 (A), Ifnb1 (B) or EMCV Vp1 (C) RNA …
Figure 7
The L region is required for IFN-α/β responses to EMCV in vivo.
(A and B) IFNAR1 KO mice were injected intraperitoneally with PBS (not infected; NI) or with 10E6 pfu of the indicated viruses. (A) mIFN-α levels in the serum were measured by ELISA after 24 hr. (B–D…
Tables
Table 1
Total number of reads aligning to the EMCV genome in LGP2 IP, ctrl IP, or input samples
| LGP2 IP | ctrl IP | input | |
|---|---|---|---|
| Number of reads* | 31,662,255 | 37,235,332 | 35,725,972 |
| EMCV | 2,747,427 | 243,472 | 1,380,128 |
| EMCV (+) | 1,305,129 | 239,625 | 1,380,058 |
| EMCV (−) | 1,442,298 | 3,847 | 70 |
-
*
Total numbers of reads, reads matching both strands (EMCV), sense strand (EMCV (+)) or antisense strand (EMCV (−)) of EMCV RNA sequences.
Table 2
Percentage of reads mapping either the sense (+) or the antisense (−) strand in the L region compared to the full length EMCV genome
| L region | Total EMCV | |||
|---|---|---|---|---|
| (+) | (−) | (+) | (−) | |
| LGP2 IP | 19.49 | 80.51 | 47.50 | 52.50 |
| ctrl IP | 78.55 | 21.45 | 98.17 | 1.83 |
| input | 100.00 | 0.00 | 100.00 | 0.00 |
