Establishing the role of ATP for the function of the RIG-I innate immune sensor.

David C Rawling,Anna Marie Pyle,Megan E Fitzgerald

doi:10.7554/elife.09391

David C Rawling, Anna Marie Pyle + Show 1 more

Open Access

https://doi.org/10.7554/elife.09391

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Journal: eLife	Publication Date: Sep 15, 2015
Citations: 93	License type: CC BY 4.0

Affiliation: Yale University, Howard Hughes Medical Institute

Abstract

Retinoic acid-inducible gene I (RIG-I) initiates a rapid innate immune response upon detection and binding to viral ribonucleic acid (RNA). This signal activation occurs only when pathogenic RNA is identified, despite the ability of RIG-I to bind endogenous RNA while surveying the cytoplasm. Here we show that ATP binding and hydrolysis by RIG-I play a key role in the identification of viral targets and the activation of signaling. Using biochemical and cell-based assays together with mutagenesis, we show that ATP binding, and not hydrolysis, is required for RIG-I signaling on viral RNA. However, we show that ATP hydrolysis does provide an important function by recycling RIG-I and promoting its dissociation from non-pathogenic RNA. This activity provides a valuable proof-reading mechanism that enhances specificity and prevents an antiviral response upon encounter with host RNA molecules.

Highlights

Retinoic acid-inducible gene I (RIG-I) is a cellular innate immune receptor that recognizes viral ribonucleic acid (RNA) in the cytoplasm and initiates a host defense response (Yoneyama et al, 2004; Ablasser et al, 2009; Baum et al, 2010)
We show that RNA-stimulated adenosine 5′-triphosphate (ATP) binding can activate RIG-I for immune signaling in the absence of hydrolysis, explaining the lack of a correlation between ATP hydrolysis and signaling
We investigate the relationship between the ATPase and immune signaling activities of RIG-I when it is bound to dsRNA ligands of varying length and composition

Summary

Introduction

Retinoic acid-inducible gene I (RIG-I) is a cellular innate immune receptor that recognizes viral ribonucleic acid (RNA) in the cytoplasm and initiates a host defense response (Yoneyama et al, 2004; Ablasser et al, 2009; Baum et al, 2010). We found that ATP binding by RIG-I on the dumbbell is qualitatively the same as that observed when bound to duplex RNA termini, and that the dumbbell is capable of stimulating ATP hydrolysis at a rate constant that is comparable to 5′ppp10L at saturating RNA concentrations (Figure 4D, Table 1). Compared to wild type and Walker A mutant constructs, where the presence of ATPγS resulted in an approximate threefold drop in affinity, ATPγS binding by the ΔCARDs construct lowered the affinity about 1.4-fold (Figure 5C, Table 1) Consistent with this observation, we detected no enhancement in the rate of RNA dissociation (koff) for ΔCARDs RIG-I in the presence of nucleotide (Figure 5D, Table 2). These results suggest a significant role for the CARDs in mediating the enhanced RNA dissociation upon nucleotide binding

Discussion

Findings

Materials and methods