Comparative analysis of viral RNA signatures on different RIG-I-like receptors
- Institut Pasteur, CNRS UMR-3569, France
- Université Paris Diderot - Paris 7, France
- Institut Pasteur, France
- Institut de Recherche Biomédicale des Armées, France
- University of Reunion Island, France
Figures
Figure 1
Rig-I Like Receptor (RLR) gene expression in stable cell lines encompassing ST-RLRs.
(A) Schematic representation of the protein domains for each RLR. Domain boundaries are indicated for human RIG-I, MDA5, and LGP2 proteins according to interpro (www.ebi.ac.uk/interpro). (B) LGP2, …
Figure 2
Efficiency of ST-RLR cells infection by negative-sense RNA virus (MV).
(A) Efficiency of Fluc (rMV2/Fluc) expressing MV replication in ST-RLR cells. ST-RLR cells were infected with the rMV2/Fluc (MOIs: 0.05, 1, 2.5). Luc activity was analyzed 5, 19, 24 and 48 hr …
Figure 3 with 1 supplement
Efficiency of ST-RLR cells infection by positive-sense RNA viruses (CHIKV).
(A) Replication efficiency of CHIKV-Rluc in ST-RLR cells. ST-RLR cells were infected with a CHIKV-Rluc (MOIs: 0.05, 1, 2.5). renLuc activity was analyzed 0, 5, 8, 10, 13, 19, 24, 32 and 40 hr …
Figure 3—figure supplement 1
Experimental approaches used to determine early and late steps of CHIKV replication.
ST-RLR cells were infected with CHIKV-Rluc or wt CHIKV at an MOI of 1. Rluc activity was measured for the CHIKV-Rluc infection. wt CHIKV-infected cells were harvested stained with an antibody …
Figure 4
Immunostimulatory activity of RNA-ligands co-purified with ST-RLRs upon infection with MV and CHIKV.
ST-RLR cells were infected with MV for 24 hr (A), CHIKV for 13 hr (B) or mock infected (C). Total cell lysate was used for total RNA purification (INPUT) and for affinity purification of RLR RNA …
Figure 5
Innate sensing of MV and CHIKV infections by different RLRs.
(A) LGP2, MDA5 and RIG-I mRNA levels in STINGshRLR cells. STING-37 reporter cell line was transduced by lentiviral vectors expressing an shRNA directed against either LGP2 (shLGP2),or MDA5 (shMDA5), …
Figure 6 with 1 supplement
NGS analysis of specific RLR viral partners purified upon infection with MV.
MDA5/RNA (A), LGP2/RNA (B) and RIG-I/RNA (C) samples were subjected to Illumina strand-specific NGS analysis. Sequencing reads were mapped to the MV genome and only the first nucleotide was …
Figure 6—figure supplement 1
NGS profile of total RNA aligned on the MV genome from ST-CH cells infected with the MV.
First position of raw read counts are plotted in a strand specific manner (light blue=Watson, dark blue=Crick strand) per position.
Figure 7 with 2 supplements
5’ copy-back DI-genome is specifically associated with RIG-I upon infection with MVΔV.
(A) RT-PCR amplification of 5’ copy-back DI-genome from cells infected with MVΔV using specific primers. Primers JM396 and JM403 were used for 5’ copy-back DI-genome amplification and JM402 and …
Figure 7—figure supplement 1
The 5’ copy-back DI genome of MVΔV.
(A) Exact sequence of the 5’ copy-back 1,236 nucleotide-long DI genome of MVΔV. Nucleotides at the position where viral polymerase resumes synthesis to transcribe the complementary 'stem' structure …
Figure 7—figure supplement 2
Comparison of immunostimulatory activities of total RNA purified from ST-RIG-I cells infected by either MV or MVΔV recombinant viruses.
100, 10, 5, 2 and 1 ng of total RNA were tranfected in STING-37 cells and Fluc activity was measured. Experiments were performed 2 times and data represents means ± SD of the technical triplicates …
Figure 8
In silico analysis of NGS data.
(A, B, C) AU content of RLR-specific RNA ligands. Number of sequenced reads (extended to 200nts) with a given AU content. Significantly enriched reads/positions are represented in orange and …
Figure 9 with 1 supplement
Analysis of purified RIG-I-specific RNA partners by NGS upon CHIKV infection.
RIG-I/RNA samples were subjected to Illumina strand-specific NGS. Sequencing reads were mapped to the CHIKV genome and only the first nucleotide was retained in the X axis. Normalization and …
Figure 9—figure supplement 1
NGS profile of total RNA aligned on the CHIKV genome from ST-CH cells infected with the CHIKV.
First position of raw read counts are plotted in a strand specific manner (ligth blue=Watson, dark blue= Crick strand) per position. The start of subgenomic RNA transcription is shown with the black …
Figure 10 with 1 supplement
qPCR analysis of specific RLR RNA signatures from MV- and CHIKV-infected cells and their immunostimulatory activity.
(A) Locations of qPCR primers on MV and CHIKV genomes. (B) MDA5 specific interaction with the N coding region upon MV infection. MDA5/RNA samples were subjected to RT-qPCR analysis with specific …
Figure 10—figure supplement 1
co-IP of MV-N mRNA on MDA5 from human monocytes.
THP1 cells were infected with MV at an MOI of 2 for 24 hr. MDA5-associated RNA molecules were obtained by co-IP. As a negative control, antibodies against a missing protein (anti-STrEP-Tag) were …
Additional files
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Supplementary file 1
Number of positions within the MV or CHIKV genomes different in total RLR RNA samples with respect to the negative control CH sample.
- https://doi.org/10.7554/eLife.11275.019
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Supplementary file 2
List of primers used for the study.
- https://doi.org/10.7554/eLife.11275.020
