Induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b,
abrogated by small-molecule ORF9b homodimerization inhibitors
Abstract
Viral accessory proteins play critical roles in viral escape form host
innate immune responses and in viral inflammatory pathogenesis. Here we
show that the SARS-CoV-2 accessory protein, ORF9b, but not other
SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7, ORF8, ORF9c,
ORF10), strongly activates inflammasome-dependent caspase-1 in A549 lung
carcinoma cells and THP-1 monocyte-macrophage cells. Exposure to
lipopolysaccharide (LPS) and ATP additively enhanced the activation of
caspase-1 by ORF9b, suggesting that ORF9b and LPS follow parallel
pathways in the activation of the inflammasome and caspase-1. Following
rational in silico approaches, we have designed small molecules
capable of inhibiting the homodimerization of ORF9b, which
experimentally inhibited ORF9b-ORF9b homotypic interactions, caused
mitochondrial eviction of ORF9b, inhibited ORF9b-induced activation of
caspase-1 in A549 and THP-1 cells, cytokine release in THP-1 cells, and
restored type I interferon (IFN-I) signaling suppressed by ORF9b in both
cell models. These small molecules are first-in-class compounds
targeting a viral accessory protein critical for viral-induced
exacerbated inflammation and escape from innate immune responses, with
the potential of mitigating the severe immunopathogenic damage induced
by highly pathogenic coronaviruses and restoring antiviral innate immune
responses curtailed by viral infection.