SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

Evolution of enhanced innate immune evasion by the SARS-CoV-2 B.1.1.7 UK variant

“…It is known that Orf9b, Orf6 and N are innate immune antagonists, acting at different levels to inhibit RNA sensing. Orf6 inhibits IRF3 and STAT1 nuclear translocation 13,24 , N prevents activation of RNA sensor RIG-I 16 and here we show that Orf9b inhibits RNA sensing through interaction with TOM70 regulated by phosphorylation. We find that B.1.1.7 has evolved to produce more sgRNA for these key innate immune antagonists leading to increased protein levels and enhanced innate immune antagonism as compared to first wave isolates.…”

“…Additionally, we detected elevated sgRNA levels in B.1.1.7 of a third innate immune regulator, nucleocapsid (N) 16 . B.1.1.7 N RNA was increased 1.7-fold and 2.3-fold compared to VIC and IC19, respectively, corresponding to a 2.4-fold and 2.3-fold increase in N protein levels (Fig.…”

“…2g). SARS-CoV-2 N has also been shown to inhibit activation of RNA sensing 16 . B.1.1.7 has a modest increase in N expression (Fig.…”

“…B.1.1.7 variant of concern (VOC)-defining mutations outside Spike suggest that Spike-independent adaptation to host may contribute to the B.1.1.7 transmission advantage. Most B.1.1.7 coding changes map to non-structural proteins Nsp3, Nsp6, accessory protein Orf8 and nucleocapsid protein (N), all of which have been shown to modulate the innate immune response [12][13][14][15][16] . Furthermore, it is unclear whether any of the B.1.1.7specific mutations impact the expression levels of viral proteins.…”

The Pathogenic Features of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): Possible Mechanisms for Immune Evasion?

“…Furthermore, the N protein is suggested to have the ability to interfere with RIG-I (43). In addition, the N protein has the ability to restrain the combination between TBK1 and IRF3, resulting in the failure of IRF3 nuclear translocation (42), while nsp12 protein just inhibits the nuclear translocation of IRF3 without interfering IRF3 phosphorylation (44).…”