ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA
- Ludwig Maximilian University of Munich, Germany
- Icahn School of Medicine at Mount Sinai, United States
- University of Bonn, Germany
- Center for Integrated Protein Science Munich, Germany
Figures
Figure 1 with 2 supplements
Cellular studies of RIG-I ATPase mutants in infected or non-infected cells.
(A) Location of amino acid substitutions of RIG-I SF2 domain variants used in this study (orange lines) within different RLR helicase motifs (orange squares). (B) Single amino acid substitutions …
Figure 1—figure supplement 1
Assay for defining of the impact of RLR variant expression on RLR signaling in infected or non-infected cells.
(A) HEK 293T RIG-I KO cells were co-transfected with different expression and control vectors as indicated. RLR signaling induces an interferon-β (IFNβ) promoter driven expression of firefly …
Figure 1—figure supplement 2
RIG-I E373Q mutation does not confer constitutive activity due to an exposed 2CARD module.
(A) Small-angle X-ray scattering (SAXS) intensity curves of RIG-I and RIG-I E373Q in presence and absence of ATP. (B) Distance distribution functions derived from SAXS data in panel A. Calculated …
Figure 2 with 3 supplements
RIG-I ATP hydrolysis defective mutant E373Q recognizes the 60S ribosomal subunit in vivo.
(A) Relative RNA amount co-purified with overexpressed RIG-I or RIG-I E373Q from virus infected or non-infected HEK 293T RIG-I KO cells. n=4 (infected) or n=10 (non-infected), error bars represent …
Figure 2—figure supplement 1
Analysis of RNA co-purified with RIG-I SMS or MDA5 variants.
(A) Relative RNA amount co-purified with overexpressed RIG-I, RIG-I E373Q or RIG-I SMS variants from non-infected HEK 293T RIG-I KO cells. (B) Relative RNA amount co-purified with overexpressed MDA5 …
Figure 2—figure supplement 2
Assay for defining the immunostimulatory potential of different RNAs.
(A) Endogenous RLRs in HEK 293T ISRE-FF/RFP cells (stably express firefly luciferase (FF) and RFP under control of an interferon stimulated response element (ISRE) promoter) induce a downstream …
Figure 2—figure supplement 3
Immunostimulatory potential of co-purified RNA from Sendai virus Cantell (SeV) infected cells.
(A) HEK 293T RIG-I KO cells were transfected with the indicated RIG-I mutant or GFP expression vector. RNA co-purified with the respective overexpressed protein was back-transfected into HEK 293T …
Figure 3
RIG-I ATP hydrolysis defective mutant E373Q recognizes the 60S ribosomal subunit in vitro.
(A) DRaCALA ATP binding assay of RIG-I or RIG-I E373Q in presence or absence of RNA. (B) ATP hydrolysis assay of RIG-I or RIG-I E373Q in presence and absence of RNA. (C) Binding studies of human 80S …
Figure 4 with 1 supplement
RIG-I’s ATP hydrolysis enhances RNA end recognition and removes RIG-I from RNA stems.
(A) Quantification of electrophoretic mobility shift assays of RIG-I or RIG-I E373Q incubated with 24mer dsRNA in presence or absence of ATP, ADP or ADP·BeF3 (compare with Figure 4—Figure supplement …
Figure 4—figure supplement 1
Design of the ribosomal expansion segment derived hairpin RNA, EMSA raw figures and control experiments with RIG-I C268F SMS mutant.
(A) RIG-I E373Q binding site at ES7L-A was used to design a 60b hairpin RNA (ES hairpin). RNA secondary structure was determined with the RNAfold webserver (Gruber et al., 2008). (B) Electrophoretic …
Figure 5 with 1 supplement
RIG-I’s ATPase activity correlates with its RNA binding affinity.
(A) Quantification of hydrolyzed [γ-32P]ATP by RIG-I or RIG-I E373Q in presence of different RNA substrates. Reactions were allowed to proceed for 20 min at 37 °C and free phosphate was separated …
Figure 5—figure supplement 1
RIG-I’s 2CARD module reduces the ATP hydrolysis activity.
(A) Measurement of ES hairpin or ppp-dsRNA stimulated [γ-32P]ATP hydrolysis of RIG-I or RIG-I Δ2CARD. Reactions were monitored over 3 hr at room temperature and free phosphate was separated from ATP …
Figure 6
Proposed model for impact of ATP on RIG-I signaling on different RNAs.
(A) RIG-I recognizes tri- or diphosphorylated double-stranded RNA and preferentially binds to the RNA end through its regulatory domain (RD, green). Binding of ATP-SF2 (purple) to the dsRNA releases …
