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

Abstract

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. Observations made for the N protein of human coronavirus 229E suggests that this may be a general trait conserved among members of other orthocoronavirus (sub)genera.

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation

“…The SARS-CoV-2 nucleocapsid, like nucleocapsids of other Betacoronaviruses, is a highly multifunctional protein. Like other coronavirus nucleocapsids, it is thought to play key roles in the packaging of viral RNA 25 , the production of viral RNA through interactions with the replication-transcription complex 10–13 , and the antagonism of the innate immune response 26–28 . To better understand why the N: G215C mutation was important at a molecular level, we looked at where in the viral life cycle the stably dimerized form of N was observed.…”

SARS-CoV-2 Variant-Specific Differences in Inhibiting the Effects of the PKR-Activated Integrated Stress Response

“…All variants triggered ISR activation, but Delta induced significantly less phosphorylation of PKR and eIF2α than the ancestral strain and Omicron. It has been suggested that the SARS-CoV-2 N protein can inhibit PKR activation by sequestering dsRNA [24, 54] and adaptations in the Delta N protein might make this process more efficient. Omicron on the other hand induces similar levels of p-PKR but increased levels of p-eIF2α, indicating stronger ISR activation.…”