LGP2 is a positive regulator of RIG-I- and MDA5-mediated antiviral responses - PubMed (original) (raw)

LGP2 is a positive regulator of RIG-I- and MDA5-mediated antiviral responses

Takashi Satoh et al. Proc Natl Acad Sci U S A. 2010.

Abstract

RNA virus infection is recognized by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), RIG-I, and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm. RLRs are comprised of N-terminal caspase-recruitment domains (CARDs) and a DExD/H-box helicase domain. The third member of the RLR family, LGP2, lacks any CARDs and was originally identified as a negative regulator of RLR signaling. In the present study, we generated mice lacking LGP2 and found that LGP2 was required for RIG-I- and MDA5-mediated antiviral responses. In particular, LGP2 was essential for type I IFN production in response to picornaviridae infection. Overexpression of the CARDs from RIG-I and MDA5 in Lgp2(-/-) fibroblasts activated the IFN-beta promoter, suggesting that LGP2 acts upstream of RIG-I and MDA5. We further examined the role of the LGP2 helicase domain by generating mice harboring a point mutation of Lys-30 to Ala (Lgp2 (K30A/K30A)) that abrogated the LGP2 ATPase activity. Lgp2 (K30A/K30A) dendritic cells showed impaired IFN-beta productions in response to various RNA viruses to extents similar to those of Lgp2(-/-) cells. Lgp2(-/-) and Lgp2 (K30A/K30A) mice were highly susceptible to encephalomyocarditis virus infection. Nevertheless, LGP2 and its ATPase activity were dispensable for the responses to synthetic RNA ligands for MDA5 and RIG-I. Taken together, the present data suggest that LGP2 facilitates viral RNA recognition by RIG-I and MDA5 through its ATPase domain.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Role of LGP2 in type I IFN production in response to various RNA viruses. (A and B) BM-derived cDCs from WT and Lgp2 −/− mice were exposed to the indicated viruses or treated with 1 μM CpG-DNA (D35) for 24 h. The concentrations of IFN-β (A) and IL-6 (B) in the culture supernatants were measured by ELISA. moi, multiplicity of infection; med, medium alone. Data are shown as means ± SD and are representative of at least three independent experiments.

Fig. 2.

Fig. 2.

Role of LGP2 in the activation of signaling pathways leading to IFN-inducible gene expression. (A) Total RNAs extracted from WT and Lgp2 −/− macrophages infected with EMCV were subjected to Northern blot analyses for the expressions of Ifnb, Cxcl10, Il6, and Actb mRNAs. (B) WT and Lgp2 −/− MEFs were infected with influenza virus followed by isolation of the total RNA. The expressions of Ifnb, Cxcl10, and Actb mRNAs were determined by Northern blot analyses. (C and D) Nuclear extracts were prepared from WT and Lgp2 −/− macrophages infected with EMCV for the indicated periods. The binding activities of DNA to NF-κB (C) and ISREs (D) were determined by EMSAs. (E) Cell lysates were prepared from WT and Lgp2 −/− macrophages infected with EMCV and probed with anti–phospho-STAT-1 and anti-STAT1 antibodies. The data are representative of at least three independent experiments.

Fig. 3.

Fig. 3.

LGP2 acts in the upstream of RIG-I and MDA5. (A) WT and Lgp2 −/− MEFs were transiently transfected with the IFN-β promoter construct together with expression plasmids encoding LGP2. The cells were infected with EMCV for 8 h and then lysed. The cell lysates were analyzed by a luciferase assay. (B) WT and Lgp2 −/− MEFs were infected with a retrovirus expressing Lgp2. At 2 days after infection, the cells were exposed to EMCV for 24 h. The IFN-β concentrations in the culture supernatants were measured by ELISA. N.D., not detected. (C) Lgp2 −/− Mda5 −/− MEFs were transiently transfected with the IFN-β promoter reporter construct together with the indicated expression plasmids. After 24 h, the cells were infected with EMCV for 8 h and then lysed. The cell lysates were analyzed by a luciferase assay. (D) Lgp2 −/− MEFs were transiently transfected with the IFN-β promoter construct together with expression plasmids encoding the CARDs of RIG-I or MDA5 and then lysed at 48 h after transfection. The cell lysates were analyzed by a luciferase assay.

Fig. 4.

Fig. 4.

Role of LGP2 in the recognition of exogenously transfected RNAs. (A) WT and Lgp2 −/− MEFs were stimulated with triphosphate RNA, in vitro–transcribed dsRNA (1 μg/mL) or poly I:C complexed with Lipofectamine 2000 for 24 h. The IFN-β concentrations in the culture supernatants were measured by ELISA. med, medium; N.D., not detected. Data are shown as the means ± SD of triplicate samples. Similar results were obtained in three independent experiments. (B) WT and Lgp2 −/− MEFs were transfected with the indicated amounts of poly I:C complexed with Lipofectamine 2000. The IFN-β concentrations in the culture supernatants were measured by ELISA.

Fig. 5.

Fig. 5.

Essential role of the LGP2 ATPase activity in the recognition of RNA viruses. (A) Lgp2 −/− MEFs were transiently transfected with the IFN-β promoter construct together with expression plasmids encoding LGP2 or LGP2 (K30A). The cells were infected with EMCV for 8 h and then lysed. The cell lysates were analyzed by a luciferase assay. (B) WT and Lgp2 −/− MEFs were infected with retroviruses expressing LGP2 or LGP2 (K30A). At 2 days after infection, the cells were exposed to EMCV for 24 h and the IFN-β concentrations in the culture supernatants were measured by ELISA. (C and D) WT and Lgp2 K30A/K30A (K30A) mice were exposed to the indicated RNA viruses for 24 h. The concentrations of IFN-β (C) and IL-6 (D) in the culture supernatants were measured by ELISA. (E) WT and Lgp2 K30A/K30A cDCs were transfected with the indicated RNAs for 24 h. The concentrations of IFN-β in the culture supernatants were measured by ELISA. moi, multiplicity of infection; med, medium alone; LF, lipofectamine alone. Data are shown as means ± SD and are representative of at least three independent experiments.

Fig. 6.

Fig. 6.

Role of LGP2 in host defense against EMCV infection in vivo. (A and B) WT and littermate Lgp2 −/− mice (A) or WT and littermate Lgp2 K30A/K30A (K30A) mice (B) were i.v. inoculated with 1 × 107 pfu EMCV. Serum samples were obtained at 4 h after injection, and the IFN-β concentrations were determined by ELISA. (C and D) Survival rates of WT and Lgp2 −/− mice (C) or WT and littermate Lgp2 K30A/K30A mice (D) intraperitoneally infected with 1 × 102 pfu EMCV were monitored every 12 h for 5 days. (E and F) WT and littermate Lgp2 −/−mice (E) or WT and littermate Lgp2 K30A/K30A mice (F) were infected i.p. with 1 × 102 pfu EMCV. After 48 h, the mice were killed and the virus titers in their hearts were determined by a plaque assay.

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