Visualizing the Determinants of Viral RNA Recognition by Innate Immune Sensor RIG-I Dahai Luo,1,4 Andrew Kohlway,2 Adriana Vela,2 and Anna Marie Pyle1,3,4,* 1Department of Molecular, Cellular, and Developmental Biology 2Department of Molecular Biophysics and Biochemistry 3Department of Chemistry Yale University, New Haven, CT 06520, USA 4Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
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*Correspondence: anna.pyle@yale.edu
http://dx.doi.org/10.1016/j.str.2012.08.029
SUMMARY
Retinoic acid inducible gene-I (RIG-I) is a key intra- cellular immune receptor for pathogenic RNAs, particularly from RNA viruses. Here, we report the crystal structure of human RIG-I bound to a 50
triphosphorylated RNA hairpin and ADP nucleotide at 2.8 Å resolution. The RNA ligand contains all structural features that are essential for optimal recognition by RIG-I, as it mimics the panhandle- like signatures within the genome of negative- stranded RNA viruses. RIG-I adopts an intermediate, semiclosed conformation in this product state of ATP hydrolysis. The structure of this complex allows us to visualize the first steps in RIG-I recognition and acti- vation upon viral infection.
INTRODUCTION
Pathogen recognition receptors (PRRs) are signaling proteins
that continually survey cells for the presence of pathogen associ-
ated molecular patterns (PAMPs). Retinoic acid inducible gene I
(RIG-I) is a major cellular PRR that senses viral RNA PAMPs in
the cytoplasm of infected cells (Kato et al., 2011; Yoneyama
et al., 2004). RIG-I recognizes a broad spectrum of viruses,
including the negative-stranded vesicular stomatitis virus, influ-
enza, and rabies viruses, and also positive-stranded viruses
such as dengue and hepatitis C virus (Kawai and Akira, 2007;
Ramos and Gale, 2011). Defective viral replication by Sendai
virus and influenza virus generates short subgenomic RNAs
that may be a principal ligand for RIG-I during viral infection
(BaumandGarcı́a-Sastre, 2011;Baumet al., 2011). At themolec-
ular level, RIG-I preferentially recognizes double stranded RNAs
that contain a triphosphate moiety at the 50 end, exemplified by thepanhandle-likeRNAsof negative-strand viruses such as influ-
enza (Hornung et al., 2006; Pichlmair et al., 2006; Schlee et al.,
2009). Recent biochemical and structural studies have shown
that the C-terminal domain (CTD) of RIG-I recognizes duplex
termini, interacting specifically with terminal 50 triphosphate moieties (Cui et al., 2008; Lu et al., 2010; Wang et al., 2010).
Structure 20, 1983–19
The central SF2 helicase domain (HEL) binds internally to the
double-stranded RNA (dsRNA) backbone (Jiang et al., 2011; Ko-
walinski et al., 2011; Luo et al., 2011). A pincer domain connects
the CTD and the HEL domains and provides mechanical support
for coordinated RNA recognition by the two domains (Luo et al.,
2011). TheN terminal tandemcaspase activation and recruitment
domains (CARDs) are responsible for downstream signaling,
leading to the expression of antiviral interferon-stimulated genes
(Jiang and Chen, 2011; Ramos and Gale, 2011).
The current model of RIG-I activation suggests that the
binding ofRNAby theHELandCTDgenerates a nanomechanical
force that releases an inhibitory conformation imposed by the
CARD domains, a process that also requires ATPase activity
through an unknown mechanism (Kowalinski et al., 2011; Luo
et al., 2011). Identifying the molecular determinants for RNA
recognition and understanding how RIG-I distinguishes viral
RNA from cellular RNA represent important unanswered ques-
tions in the field of innate immunity. Here, we report the crystal
structure of RIG-I in complex with a 50 triphosphorylated double-stranded RNA and adenosine nucleotide, thereby
providing the biologically relevant snapshot of viral PAMP recog-
nition by RIG-I. We show that binding of different ATP analogs
induces specific conformational changes within the protein,
verifying the structural observations and supporting a tightly
regulated, multistep activation mechanism of RIG-I.
RESULTS AND DISCUSSION
To unravel the molecular details of viral PAMP recognition by
RIG-I, we designed a hairpin RNA (hereafter named as 50
ppp8L which contains a 50 triphosphate moiety and a stem of 8 base pairs that is terminated by a UUCG tetra loop) that mimics
the panhandle-like genome of negative-stranded RNA viruses
(Figures S1 and S2 available online). We cocrystallized 50
ppp8L with a human RIG-I construct that lacks the CARD
domains (RIG-I [DCARDs: 1–238]; Figure 1). All atoms of the
RNA hairpin are observed and unambiguously built into the
2.8 Å density map (Figure 1C; Table 1).
The overall structure of the complex (RIG-I (DCARDs: 1–238):
50 ppp8L: ADP-Mg2+) is similar to the RIG-I:dsRNA10 structure reported previously (rmsd = 0.38 Å for 559 superimposed Ca
atoms) (Luo et al., 2011). However, in the structure reported
88, November 7, 2012 ª2012 Elsevier Ltd All rights reserved 1983
mailto:anna.pyle@yale.edu
http://dx.doi.org/10.1016/j.str.2012.08.029
Figure 1. Ternary Complex of RIG-I
(DCARDs 1–238): 50 ppp8L: ADP-Mg2+
(A) Structure of the 50 triphosphorylated hairpin RNA (50 ppp8L, in purple with 50 GTP in red) bound at the center of the RIG-I (DCARDs). Bound ADP-
Mg2+ is in purple.
(B) The 50 triphosphate binding site at CTD. Fo-Fc omit map is in green and contoured at 3.5 s.
(C) Superposition of RIG-I with 50 triphosphory- lated hairpin RNA and RIG-I with 50 hydroxyl dsRNA in gray (PDB: 2ykg).
See also Figures S1 and S2.
Structure
Structure of RIG-I, 50 ppp-dsRNA, and ADP
here, the CTD encapsulates the 50 triphosphate moiety at the duplex terminus. Functional groups along the RNA duplex
interact with the HEL1 and HEL2i domains as observed
previously. Importantly, one can now observe the position of
bound nucleotide, revealing that ADP interacts exclusively
with conserved ATPase motifs localized in HEL1 (Figure 1A).
HEL2 is not involved in RNA binding or ADP binding (Figure 1).
The protein conformation observed in this structure is likely to
be biologically relevant because we observe that 50 ppp8L RNA readily stimulates efficient ATP hydrolysis by RIG-I (Fig-
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The post Visualizing the Determinants of Viral RNA Recognition by Innate Immune Sensor RIG-I Dahai Luo,1,4 Andrew Kohlway,2 Adriana Vela,2 and Anna Marie Pyle1,3,4,* 1Department of Molecular, Cellular, and Developmental Biology 2Department of Molecular Biophysics and Biochemistry 3Department of Chemistry Yale University, New Haven, CT 06520, USA 4Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA appeared first on My Nursing Papers.
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