Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling

Abstract

The oligoadenylate synthetase (OAS) enzymes are cytoplasmic dsRNA sensors belonging to the antiviral innate immune system. Upon binding to viral dsRNA, the OAS enzymes synthesize 2'-5' linked oligoadenylates (2-5As) that initiate an RNA decay pathway to impair viral replication. The human OAS-like (OASL) protein, however, does not harbor the catalytic activity required for synthesizing 2-5As and differs from the other human OAS family members by having two C-terminal ubiquitin-like domains. In spite of its lack of enzymatic activity, human OASL possesses antiviral activity. It was recently demonstrated that the ubiquitin-like domains of OASL could substitute for K63-linked poly-ubiquitin and interact with the CARDs of RIG-I and thereby enhance RIG-I signaling. However, the role of the OAS-like domain of OASL remains unclear. Here we present the crystal structure of the OAS-like domain, which shows a striking similarity with activated OAS1. Furthermore, the structure of the OAS-like domain shows that OASL has a dsRNA binding groove. We demonstrate that the OAS-like domain can bind dsRNA and that mutating key residues in the dsRNA binding site is detrimental to the RIG-I signaling enhancement. Hence, binding to dsRNA is an important feature of OASL that is required for enhancing RIG-I signaling.

Figure 1.

Full-length OASL forms soluble multimers while the OLD is a monomer. (A) Schematic representation of the domain organization of full-length OASL, the OLD and the UbLD. (B) Chromatograms of Superdex 200 PC3.2/30 analytical size exclusion chromatography of OASL, Blue Dextran and albumin in solid, dashed and dotted lines, respectively. ‘Multimer’ and ‘monomer’ indicate OASL elution peaks for multimeric and monomeric OASL, respectively. (C) A Coomassie stained 8% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the following: load: OASL loaded on the size exclusion chromatography column. Multimer: The OASL eluted in the void volume at ‘multimer’ as shown in (B). Monomer: Monomeric OASL eluted at ‘monomer’ as shown in (B). (D) Chromatograms of Superdex 75 HiLoad 16/60 size exclusion chromatography of OLD, β-amylase, albumin and carbonic anhydrase in solid, dashed, dotted and interchanging lines, respectively. (E) A Coomassie stained 10% SDS-PAGE analysis of retention volumes from the size exclusion chromatography shown in (D).

Figure 2.

The structure of the OLD is remarkably similar to the activated form of OAS1. (A) Model building in the electron density contoured at 2σ. The displayed model and electron density include the αN4 helix. The side chains of Val67, Phe70 and Asn72 are indicated. (B) The crystal structure of OLD. The N-terminal extension and the linker regions that connect the N- and C-terminal lobes are indicated and colored yellow–green. The αN3, αN4 and αN5 helices are indicated and colored green. The five-stranded antiparallel β-sheet is indicated and colored dark green. The C-terminal lobe is indicated and colored light green. (C) Superposition of the OLD (green, PDB: 4XQ7) on active OAS1 (human OAS1 in complex with dsRNA, yellow, PDB: 4IG8) and inactive OAS1 (porcine OAS1, magenta, PDB: 1PX5). A zoom-in on the αN4 present in the active OAS1 and OLD but not in the inactive OAS1. The dashed line in the inactive OAS1 indicates the unstructured residues forming the αN4. (D) Superposition as in (C) showing the position of the αN3 and αN5 helices. The arrows indicate the RNA-induced rearrangement of αN3 and αN5 in OAS1.

Figure 3.

Shared and unique secondary structure features of OLD (PDB: 4XQ7) and active and inactive OAS1. The secondary structure is presented under the alignment and displays the shared secondary structure unless otherwise indicated. The structure of pOAS1 (PDB: 1PX5) was determined in the apo-form and thus represents the inactive OAS1 conformation. The structure of hOAS1 (PDB: 4IG8) was determined in complex with dsRNA and thus represents the active OAS1 conformation. α-helices are depicted as red waves and labeled according to position in the N-terminal (N) or C-terminal (C) lobe. β-sheets are depicted as yellow arrows and labeled according to overall position. Asterisks mark the catalytic triad of OAS1. Daggers mark residues important for binding to dsRNA. Dotted lines indicate disordered regions. SS: secondary structure.

Figure 4.

The αN4 is an integral part of OASL. (A) The crystal structure of OLD with a zoom-in on the αN4 helix. The anchoring residues Val67, Phe70 and Asn72 are indicated and their side chains are shown in sticks. (B) An alignment of porcine OAS1 (pOAS1), human OAS1 (hOAS1), human cGAS (hcGAS), human OASL (hOASL), Archaeoglobus fulgidus CCA (AfCCA) and Saccharomyces cerevisiae PAP (yPAP). The residues spanning the αN4 or the αN4-equivalent helices in cGAS, CCA and PAP are indicated. The asterisks indicate the three carboxyl acid residues in the catalytic triad of OAS1, cGAS, CCA and PAP. The residues are colored according to conservation. (C) HEK293T OASL^neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL V67G or OASL N72K in combination with an IFN-β promoter firefly luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. (D) HeLa cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL V67G or OASL N72K in combination with an IFN-β promoter firefly luciferase reporter construct and a β-actin promoter Renilla luciferase construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. The measurements are shown as a ratio of firefly to Renilla luciferase activities. Error bars indicate standard deviation from three independent experiments. **: P-value ≤ 0.01. ***: P-value ≤ 0.001.

Figure 5.

OASL has a dsRNA binding groove. (A) Surface charge representation of human OAS1 in complex with dsRNA (PDB: 4IG8). (B) Surface charge representation of the OLD (PDB: 4XQ7). Residues in the dsRNA binding groove that are mutated in Figure 6E are indicated at their position. The surface charge colors range from +5 to -5 k_bT/e_c.

Figure 6.

OASL binds to dsRNA and dsRNA binding is essential to enhance RIG-I signaling. (A) A constant concentration of OLD was incubated with increasing amounts of 18 bp dsRNA and subsequently analyzed by silver stained native PAGE. OLD was kept constant at 2.4 μM (1 μg) and incubated with 0, 0.13, 0.27, 0.54, 1.1, 2.2, 4.3 or 8.6 μM (1 μg) 18 bp dsRNA. The negative control was performed with 8.6 μM 18 bp dsRNA and no OLD. (B) 2.4 μM wild-type OLD or K66E OLD was incubated with 8.6 μM 18 bp dsRNA and analyzed by native PAGE. The proteins were visualized by silver staining or immunoblotting against the 6xHis-tag. Wells: the position of the bottom of the wells in the corresponding native gel. (C) A constant concentration of 18 bp dsRNA was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of 18 bp dsRNA was kept constant at 10 μM and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. (D) A constant concentration of poly(I:C)-LMW was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of poly(I:C)-LMW was kept constant at 0.2 mg/ml and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. (E) HEK293T OASL^neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL R45E/K66E/R196E/K200E (RKRK), OASL K63E, OASL K66E or OASL S327A in combination with an IFN-β promoter luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. Ns: P-value > 0.05. ***: P-value ≤ 0.001.