SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b

• Published in: PLoS pathogens Vol. 19; no. 8; p. e1011582
• Main Authors: Aloise, Chiara, Schipper, Jelle G., van Vliet, Arno, Oymans, Judith, Donselaar, Tim, Hurdiss, Daniel L., de Groot, Raoul J., van Kuppeveld, Frank J. M.
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 22.08.2023; Public Library of Science (PLoS)
• Subjects: Analysis; Antiviral drugs; Binding; Biological response modifiers; Biology and life sciences; Coronaviruses; COVID-19; Double-stranded RNA; Genomes; Innate immunity; Interferon; Kinases; Medicine and health sciences; Microscopy; Middle East respiratory syndrome; Mode of action; N protein; Nucleocapsids; Packaging; Plasmids; Post-translation; Proteins; Research and Analysis Methods; Respiratory diseases; Sensors; Sequestering; Severe acute respiratory syndrome coronavirus 2; Stress response; Viral diseases; Viral infections; Viruses; β-Interferon; Netherlands
• DOI: 10.1371/journal.ppat.1011582
• ISSN: 1553-7374; 1553-7366

The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding—prevented PKR activation, inhibited β-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera.