SARS-CoV-2 ORF3a-Mediated NF-κB Activation Is Not Dependent on TRAF-Binding Sequence

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a global pandemic of Coronavirus Disease 2019 (COVID-19). Excessive inflammation is a hallmark of severe COVID-19, and several proteins encoded in the SARS-CoV-2 genome are capable of stimulating inflammatory pathways. Among these, the accessory protein open reading frame 3a (ORF3a) has been implicated in COVID-19 pathology. Here we investigated the roles of ORF3a in binding to TNF receptor-associated factor (TRAF) proteins and inducing nuclear factor kappa B (NF-κB) activation. X-ray crystallography and a fluorescence polarization assay revealed low-affinity binding between an ORF3a N-terminal peptide and TRAFs, and a dual-luciferase assay demonstrated NF-κB activation by ORF3a. Nonetheless, mutation of the N-terminal TRAF-binding sequence PIQAS in ORF3a did not significantly diminish NF-κB activation in our assay. Our results thus suggest that the SARS-CoV-2 protein may activate NF-κB through alternative mechanisms.

Keywords: NF-κB; SARS-CoV-2 ORF3a; TRAF proteins; inflammation.

Figure 1

TRAF2 and TRAF3 TRAF-C domains in complex with ORF3a peptide. (A) Trimer structure of TRAF2 TRAF-C (upper) or TRAF3 TRAF-C (lower) in complex with one or two ORF3a peptides, respectively. Five peptide residues, PIQAS, are shown as orange or green ball-and-stick models in the TRAF2 and TRAF3 structures, respectively. (B) Fo-Fc omit map (blue mesh, 2.5 sigma) in the peptide-binding groove of TRAF2 (upper panel) or TRAF3 (lower panel) with PIQAS residues from the ORF3a peptide superimposed. (C) Residues from selenomethionine (Se-Met)-containing ORF3a peptide in complex with the TRAF2 TRAF-C domain. Anomalous difference map (magenta mesh) is shown at 4 sigma. (D) Hydrogen bonds between residues in ORF3a peptide and residues in TRAF2 (left) or TRAF3 (right) TRAF-C domains are shown as gray dotted lines.

Figure 2

Fluorescence polarization (FP) assay with 5-FAM-ORF3a peptide and TRAF2 or TRAF3 TRAF-C domain. (A) FP assay with 5-FAM-ORF3a peptide alone (0–256 nM) to determine optimum concentrations for experiments with TRAF proteins. (B) Nonlinear regression curves generated from FP assays. Left: Binding of 0–256 μM of TRAF2 TRAF-C domain and 30 nM of 5-FAM-ORF3a peptide. Two independent experiments conducted in triplicate. Right: FP assay with 0–96 μM of TRAF3 TRAF-C domain and 50 nM of 5-FAM-ORF3a peptide. Three independent experiments conducted in triplicate. Plots generated and statistics calculated in GraphPad Prism. The error bars represent standard deviation (SD).

Figure 3

SARS-CoV-2 ORF3a-mediated NF-κB activation. (A) HEK293T cells were transfected with NF-κB responsive firefly luciferase plasmid and constitutive Renilla luciferase plasmid, plus empty vector or increasing amounts of SARS-CoV-2 ORF3a-expressing plasmid (1.7, 3.3, or 5 μg per well in 12-well plates). Lysates were harvested 48 h after transfection and used in a dual-luciferase assay. The ratio of firefly to Renilla luciferase activity was plotted. As a positive control, cells were treated with 5 ng/mL of TNF-⍺ for 6 h prior to harvesting. (B) SARS-CoV-2 ORF3a in HEK293T dual-luciferase samples was detected by immunoblotting with anti-SARS-CoV-2 ORF3a antibody. Detection of GAPDH was used as a loading control. (C) HEK293T cells were transfected with NF-κB responsive firefly luciferase plasmid and constitutive Renilla luciferase plasmid, plus empty vector or plasmids expressing wildtype or TRAF-mut ORF3a. Lysates were harvested 48 h after transfection and assays were performed similar to (A). p-values: empty vs. ORF3a = 0.0017, ORF3a vs. ORF3a TRAF-mut (ns) = 0.145. ** p < 0.01. Plots were generated and statistics were calculated in GraphPad Prism. The error bars represent standard deviation (SD).