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. 2013 Jan 11;288(2):938-46.
doi: 10.1074/jbc.M112.424416. Epub 2012 Nov 26.

ATP hydrolysis enhances RNA recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology 2 (LGP2)

Annie M Bruns  1 , Darja Pollpeter, Nastaran Hadizadeh, Sua Myong, John F Marko, Curt M Horvath
Affiliations

ATP hydrolysis enhances RNA recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology 2 (LGP2)

Annie M Bruns et al. J Biol Chem. .

Abstract

Laboratory of genetics and physiology 2 (LGP2) is a member of the RIG-I-like receptor family of cytoplasmic pattern recognition receptors that detect molecular signatures of virus infection and initiate antiviral signal transduction cascades. The ATP hydrolysis activity of LGP2 is essential for antiviral signaling, but it has been unclear how the enzymatic properties of LGP2 regulate its biological response. Quantitative analysis of the dsRNA binding and enzymatic activities of LGP2 revealed high dsRNA-independent ATP hydrolysis activity. Biochemical assays and single-molecule analysis of LGP2 and mutant variants that dissociate basal from dsRNA-stimulated ATP hydrolysis demonstrate that LGP2 utilizes basal ATP hydrolysis to enhance and diversify its RNA recognition capacity, enabling the protein to associate with intrinsically poor substrates. This property is required for LGP2 to synergize with another RIG-I-like receptor, MDA5, to potentiate IFNβ transcription in vivo during infection with encephalomyocarditis virus or transfection with poly(I:C). These results demonstrate previously unrecognized properties of LGP2 ATP hydrolysis and RNA interaction and provide a mechanistic basis for a positive regulatory role for LGP2 in antiviral signaling.

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Figures

FIGURE 1.
FIGURE 1.
ATP enhances LGP2 interaction with dsRNA. A, the ATP hydrolysis assay demonstrates dsRNA-induced activity for RIG-I (white bars), but basal and dsRNA-induced activity for LGP2 at varying concentrations of poly(I:C) (black bars). The addition of the nonhydrolyzable analogues AMP-PNP and ADP-AlF4 to the assay inhibits the ATP hydrolysis activity of LGP2 (gray bars). * = p < 0.05. B, quantitation of dsRNA molecules bound by LGP2 (80 nm) in the absence and presence of 50 or 500 μm ATP. ATP increases the percentage of dsRNA substrates (traces) that exhibit binding by LGP2. * = p < 0.05; ** = p < 0.008. C, quantification of the number of binding and unbinding events by LGP2 per dsRNA molecule (trace) in the absence and presence of 500 μm ATP over a 120-s (1200 frame) image capture. ATP increases the number of events per RNA molecule. * = p < 0.05; ** = p < 0.02; *** = p < 0.0003. D, percent of dsRNA molecules bound by LGP2 (80 nm) in the absence and presence of ATP (black bar), AMP-PNP, ADP-AlF4, (white bars) or equimolar mixtures of ATP and analogue (gray bars). Values presented are the average of at least two independent experiments, and error bars represent S.D. * = p < 0.02; ** = p < 0.003; *** = p < 0.0003.
FIGURE 2.
FIGURE 2.
LGP2 basal ATP hydrolysis, but not dsRNA-stimulated ATP hydrolysis, is required for enhanced dsRNA recognition. A, diagram representing the domain structure of LGP2 and illustrating mutations. B, rate of ATP hydrolysis by LGP2 and mutants in the absence (black bars) or presence of 5 μg/ml of poly(I:C) (white bars). All mutants lack basal ATP hydrolysis activity, however, the presence of poly(I:C) stimulates LGP2 MIIa and K651E to hydrolyze ATP near wild type levels. * = p < 0.003; ** = p < 0.0001. C, analysis of ATP-enhanced RNA recognition by LGP2 and mutants, represented as the percent increase in the number of dsRNA molecules with binding events after addition of 500 μm ATP, compared with protein without ATP added. LGP2 displays an average 30% increase in the number of molecules bound with the addition of ATP, but no mutants display increased recognition in the presence of ATP. * = p < 0.003.
FIGURE 3.
FIGURE 3.
ATP enables LGP2 to associate with diverse imperfect dsRNAs. A, diagram illustrates the standard dsRNA and a series of increasingly noncomplementary dsRNA substrates tested. The Kd measured in the absence of ATP at 80 nm protein concentration is indicated below with S.D. in parentheses. B, representative experimental data demonstrating the percent of dsRNA molecules with binding events at 80 nm LGP2 in the absence and presence of 500 μm ATP. The dsRNA substrate and 3 top bulge substrate were evaluated in parallel chambers of the same flow cell. C, analysis of ATP-enhanced RNA recognition by 80 nm LGP2 in the presence of 500 μm ATP. Bulged substrate recognition is greatly enhanced by ATP. For each experiment, the percentage of dsRNA molecules recognized by LGP2 in the absence of ATP is set equal to 1 for comparison. Fewer bulged substrates are recognized in the absence of ATP, but in the presence of ATP similar numbers of bulged and perfectly complementary substrates are bound by LGP2. * = p < 0.05.
FIGURE 4.
FIGURE 4.
LGP2 ATP hydrolysis is required for enhanced MDA5-mediated IFNβ signaling. A, HEK293T cells were transfected with a −110 IFNβ-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5 or LGP2. MDA5 was transfected at a constant 25 ng of plasmid/well, whereas the amount of LGP2 transfected varied at 0.03, 0.16, 0.8, 4, 20, 100, and 500 ng. Following a 24-h transfection, cells were transfected with 5 μg/ml of poly(I:C) (left) or infected with 3 pfu/cell EMCV (right) for 8 h before harvesting. At low concentrations LGP2 enhances MDA5-mediated IFNβ signaling, but at higher concentrations LGP2 functions as a negative regulator. B, HEK293T cells were transfected with a −110 IFNβ-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5, LGP2 MI, or LGP2 MIIa mutants. MDA5 was transfected at a constant 25 ng of plasmid/well, whereas the amount of LGP2 transfected varied at 0.03, 0.16, 0.8, 4, 20, 100, and 500 ng. Following a 24-h transfection, cells were transfected with 5 μg/ml of poly(I:C) for 8 h before harvesting. Neither LGP2 MI nor LGP2 MIIa are able to enhance MDA5-mediated signaling. C, HEK293T cells were transfected with a −110 IFNβ-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5, LGP2, or LGP2 mutants. MDA5 was transfected at a constant 50 ng of plasmid/well, whereas the amount of LGP2 transfected was varied at 5 (white), 10 (gray), and 50 ng (black) of plasmid. Following a 24-h transfection, cells were infected with 3 pfu/cell EMCV or transfected with 5 μg/ml of poly(I:C) for 8 h before harvesting. None of the ATP hydrolysis defective mutants are able to enhance MDA5-mediated signaling.
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