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. 2013 May 9;8(5):e64202.
doi: 10.1371/journal.pone.0064202. Print 2013.

LGP2 plays a critical role in sensitizing mda-5 to activation by double-stranded RNA

Kay S Childs  1 , Richard E Randall, Stephen Goodbourn
Affiliations

LGP2 plays a critical role in sensitizing mda-5 to activation by double-stranded RNA

Kay S Childs et al. PLoS One. .

Abstract

The DExD/H box RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene-5 (mda-5) sense viral RNA in the cytoplasm of infected cells and activate signal transduction pathways that trigger the production of type I interferons (IFNs). Laboratory of genetics and physiology 2 (LGP2) is thought to influence IFN production by regulating the activity of RIG-I and mda-5, although its mechanism of action is not known and its function is controversial. Here we show that expression of LGP2 potentiates IFN induction by polyinosinic-polycytidylic acid [poly(I:C)], commonly used as a synthetic mimic of viral dsRNA, and that this is particularly significant at limited levels of the inducer. The observed enhancement is mediated through co-operation with mda-5, which depends upon LGP2 for maximal activation in response to poly(I:C). This co-operation is dependent upon dsRNA binding by LGP2, and the presence of helicase domain IV, both of which are required for LGP2 to interact with mda-5. In contrast, although RIG-I can also be activated by poly(I:C), LGP2 does not have the ability to enhance IFN induction by RIG-I, and instead acts as an inhibitor of RIG-I-dependent poly(I:C) signaling. Thus the level of LGP2 expression is a critical factor in determining the cellular sensitivity to induction by dsRNA, and this may be important for rapid activation of the IFN response at early times post-infection when the levels of inducer are low.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. LGP2 enhances IFN induction in response to poly(I:C).
(A–C) HEK293 cells were transfected with a reporter plasmid containing the luciferase gene under the control of the IFN-β promoter, a plasmid constitutively expressing β-galactosidase as a transfection control, and (A) 0.4 ng plasmids expressing mda-5 or RIG-I, (B) 100 ng plasmid expressing LGP2, or (C) 0–160 ng plasmid expressing LGP2. Total amounts of DNA were kept constant by supplementing with the empty vector pEFpl2. 24 hours after transfection cells were further transfected with the indicated amounts of poly(I:C) for 16 hours. Cell lysates were analysed for luciferase and β-galactosidase activity, and relative expression levels calculated. The effect of LGP2 on induction by poly(I:C) is statistically significant (p<0.01).
Figure 2
Figure 2. LGP2 stimulation of poly(I:C) signaling is dependent upon endogenous mda-5.
HEK293 cells were transfected with the IFN-β reporter plasmid, the β-galactosidase expression plasmid, either the empty vector pEFpl2 or pEF.LGP2, and either a control siRNA (A), an siRNA directed against RIG-I (B), or an siRNA directed against mda-5 (C). 24 hours after transfection cells were further transfected with the indicated amounts of poly(I:C) for 16 hours. Cell lysates were analysed for luciferase and β-galactosidase activity, and relative expression levels calculated. (D) The effectiveness of siRNAs against RIG-I and mda-5 was tested by immunoblotting. HEK293 cells were transfected with a vector expressing Flag-tagged RIG-I and either a control siRNA or the RIG-I siRNA (upper panel). Cells transfected with either the control siRNA or the mda-5 siRNA were exposed to IFN for 16hrs to induce mda-5 expression (lower panel). Cell extracts were subjected to immunoblotting with either anti-Flag (for RIG-I detection), anti-mda-5 or anti-tubulin as a loading control.
Figure 3
Figure 3. LGP2 co-operates with mda-5 but not RIG-I to promote poly(I:C) signaling.
HEK293 cells were transfected with the IFN-β reporter plasmid, the β-galactosidase expression plasmid, and combinations of plasmids expressing mda-5 (0.4 ng), RIG-I (0.4 ng) or LGP2 (2 ng). 24 hours after transfection cells were further transfected with increasing amounts of poly(I:C) for 16 hours. Cell lysates were analysed for luciferase and β-galactosidase activity, and relative expression levels calculated.
Figure 4
Figure 4. LGP2 knockdown cells induce less IFN in response to poly(I:C) than wild-type cells.
(A) Extracts from parental HEK293 cells and stable cell lines expressing an shRNA against LGP2 were subjected to western blotting with antibodies against LGP2 or α-tubulin as a loading control. (B) Parental HEK293 cells and LGP2 knockdown cells were transfected with the IFN-β reporter plasmid and the β-galactosidase expression plasmid. 24 hours after transfection cells were further transfected with poly(I:C) for 16 hours. (C) Parental HEK293 cells and LGP2 knockdown cells were transfected with the IFN-β reporter plasmid, the β-galactosidase expression plasmid, and either the empty vector pEFpl2 or pEF.LGP2. 24 hours after transfection cells were further transfected with poly(I:C) for 16 hours where indicated. (D) Parental HEK293 cells and LGP2 knockdown cells were transfected with the IFN-β reporter plasmid, the β-galactosidase expression plasmid, and plasmids expressing mda-5, LGP2 or IPS-1. (E) HEK293 cells were transfected with the IFN-β reporter plasmid, the β-galactosidase expression plasmid and plasmids expressing IPS-1, the CARD domains of mda-5 or LGP2 as indicated. Cell lysates were analysed for luciferase and β-galactosidase activity, and relative expression levels calculated.
Figure 5
Figure 5. Analysis of the regions of LGP2 required for stimulation of poly(I:C) signaling.
(A) Schematic diagram of LGP2 mutants. (B–E) HEK293 cells or LGP2 knockdown cells were transfected with the IFN-β reporter plasmid, the β-galactosidase expression plasmid, and plasmids expressing mda-5, RIG-I, LGP2, or various mutants of LGP2. 24 hours after transfection cells were further transfected with poly(I:C) for 16 hours where indicated. Cell lysates were analysed for luciferase and β-galactosidase activity, and relative expression levels calculated. The average of three independent experiments is shown. (F) HEK293 cells were transfected with plasmids expressing Flag-tagged mutants of LGP2. Lysates of transfected cells were subjected to western blotting using an anti-Flag antibody to confirm expression.
Figure 6
Figure 6. LGP2 interacts with mda-5 in a dsRNA-dependent manner.
(A) HEK293 cells were transfected with a plasmid expressing the helicase domain of mda-5 with a Flag tag (Flag.MH) and a plasmid expressing LGP2 with a V5 tag (V5.LGP2). 24 hours after transfection, cells were transfected with poly(I:C) for the indicated times. Cell extracts were subjected to immunoprecipitation with the anti-flag antibody, and proteins present in the precipitate were analysed by western blotting with anti-V5 and anti-flag antibodies. (B) Yeast cells were transformed with a plasmid expressing the helicase domain of mda-5 as a GAL4DBD fusion, and a plasmid expressing LGP2 or the indicated mutants of LGP2 as a GALAD fusion. Positive transformants were selected on SD-L-W, and growth on this media indicates that the yeast have been transformed by both plasmids. They were then streaked onto SD-L-W-H + 2 mM 3-AT and growth on this media demonstrates an interaction between the GAL4DBD fusion protein and the GAL4AD fusion protein. (C) Yeast cells were transformed with a plasmid expressing the helicase domain of mda-5 as a GAL4DBD fusion, a plasmid expressing LGP2 as a GAL4AD fusion and either the empty vector pHON7 (−), pHON7 expressing the dsRNA binding domains of PKR (PKR(1–207)) or pHON7 expressing a mutant form of PKR that is unable to bind dsRNA (M2(1–207)). Positive transformants were selected on SD-L-W-U, and growth on this media indicates that the yeast have been transformed by all three plasmids. They were then streaked onto SD-L-W-U-H + 2 mM 3-AT and growth on this media demonstrates an interaction between the GAL4DBD fusion protein and the GAL4AD fusion protein.
Figure 7
Figure 7. PIV5-V blocks the interaction between mda-5 and LGP2 to inhibit IFN induction.
(A) Yeast cells were transformed with a plasmid expressing the helicase domain of mda-5 as a GAL4DBD fusion, a plasmid expressing LGP2 as a GAL4AD fusion and either the empty vector pHON7 (−) or pHON7 expressing the V protein from PIV5 (PIV5-V). Positive transformants were selected on SD-L-W-U and subsequently streaked onto SD-L-W-U-H + 5 mM 3-AT to assay for an interaction between the GAL4DBD fusion protein and the GAL4AD fusion protein. (B) HEK293 cells were transfected with the IFN-β reporter plasmid, the β-galactosidase expression plasmid and either the empty vector pEFpl2 (−), pEF.LGP2 or pEF.PIV5-V as indicated. 24 hours after transfection cells were further transfected with 2 ng poly(I:C) for 16 hours. Cell lysates were analysed for luciferase and β-galactosidase activity, and relative expression levels calculated.
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