Cytosolic viral sensor RIG-I is a 5'-triphosphate-dependent translocase on double-stranded RNA

Abstract

Retinoic acid inducible-gene I (RIG-I) is a cytosolic multidomain protein that detects viral RNA and elicits an antiviral immune response. Two N-terminal caspase activation and recruitment domains (CARDs) transmit the signal, and the regulatory domain prevents signaling in the absence of viral RNA. 5'-triphosphate and double-stranded RNA (dsRNA) are two molecular patterns that enable RIG-I to discriminate pathogenic from self-RNA. However, the function of the DExH box helicase domain that is also required for activity is less clear. Using single-molecule protein-induced fluorescence enhancement, we discovered a robust adenosine 5'-triphosphate-powered dsRNA translocation activity of RIG-I. The CARDs dramatically suppress translocation in the absence of 5'-triphosphate, and the activation by 5'-triphosphate triggers RIG-I to translocate preferentially on dsRNA in cis. This functional integration of two RNA molecular patterns may provide a means to specifically sense and counteract replicating viruses.

Fig. 1. PIFE visualization of RIG-I binding and translocation

A. dsRNA (25mer) with single fluorophore (DY547) was tethered to surface via biotin-neutravidin. B. Addition of RIGh (CARD-less mutant) resulted in an abrupt increase in emission of the fluorophore, indicating RIGh binding due to PIFE (protein induced fluorescence enhancement). C, D. Addition of RIGh with ATP induced periodic fluctuation of fluorophore. E. The effect of PIFE is visible on the single molecule imaging surface i.e. fluorescence become substantially brighter upon adding RIGh protein. F. Schematic representation of three RIG-I variants used in this study; wtRIG (RIG-I wild type), RIGh (CARD-less RIG-I) and svRIG (RIG-I splice variant) is shown.

Fig. 2. RIG-I translocates on dsRNA and CARD is inhibitory

A, C, E. 100nM RIGh (CARD-less mutant), wtRIG (RIG-I wild type) and svRIG (splice variant RIG-I) was added with 1mM ATP respectively at 37°C. The signal fluctuation represents RIG-I movement along dsRNA substrate. B, D, F. Dwell time analysis of periods denoted by Δt with a double-arrow (C) was measured for many molecules and plotted as histograms for RIGh (B), wtRIG (D) and svRIG (F) for 25bp and 40bp dsRNA. G. The inverse of average Δt, (t_avg)⁻¹, was plotted against [ATP] axis and fitted to Michaelis-Menten equation. The error bars denote standard deviation from three separate experiments each. The V_max and K_m values were 0.91 and 179μM respectively.

Fig. 3. 5′-triphosphate accelerates RIG-I translocation activity

A. The translocation assay was performed on 5′-triphosphate containing substrate which consists of 86mer ssRNA from in vitro transcription annealed to complementary 20mer ssDNA with 3′ Cy3 and 5′ biotin. B, D. Activity of wtRIG was greatly stimulated by the presence of 5′-triphosphate. The data shown was taken at room temperature due to extremely fast kinetic rate observed at 37°C. RIGh also showed robust translocation activity on this substrate. C, E. Dwell time analysis indicates that wtRIG showed slightly higher rate of translocation than RIGh.

Fig. 4. RIG-I translocates on double strand of 5′-triphosphate RNA

A. To identify the region of translocation, the double strand length was varied from 20bp to 50bp while ssRNA length was varied from 66nt to 36nt respectively. B. t_avg vs duplex length. C. Three constructs were prepared with the fixed double strand RNA length of 25bp and variable ssRNA tail length of 0, 10 and 25nt. D. t_avg vs ssRNA tail length. Error bars denote standard deviation from three separate experiments each. E. Proposed mechanism for pathogen associated molecular pattern (PAMP) signal integration by RIG-I. Binding of RIG-I regulatory domain (pink) to RNA 5′ triphosphates, e.g. arising from virus replication or transcription, dimerizes RIG-I and activates the translocase domain (blue). Further recognition of dsRNA stimulates ATPase activity resulting in translocation (red arrow), a process that may induce a signaling conformation with exposed CARDs (gray). Precise domain arrangements are speculative and depicted here for illustrative purposes.