SARS-CoV-2 Nsp14 binds Tollip and activates pro-inflammatory pathways while downregulating interferon-α and interferon-γ receptors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 14 (Nsp14) possesses an N-terminal exonuclease (ExoN) domain that provides a proofreading function for the viral RNA-dependent RNA polymerase and a C-terminal N7-methyltransferase (N7-MTase) domain that methylates viral mRNA caps. Nsp14 also modulates host functions. This includes the activation of NF-κB and downregulation of interferon alpha/beta receptor 1 (IFNAR1). Here, we demonstrate that Nsp14 exerts broader effects, activating not only NF-κB responses but also extracellular-signal-regulated kinase (ERK), p38, and Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling, promoting cytokine production. Furthermore, Nsp14 downregulates not only IFNAR1 but also IFN-γ receptor 1 (IFNGR1), impairing cellular responses to both IFNα and IFNγ. IFNAR1 and IFNGR1 downregulation is via a lysosomal pathway and occurs in SARS-CoV-2-infected cells. Analysis of a panel of Nsp14 mutants reveals a consistent pattern. Mutants that disable ExoN function remain largely active, whereas N7-MTase mutations impair both pro-inflammatory pathway activation and IFN receptor downregulation. Innate immune modulating functions also require the presence of both the ExoN and N7-MTase domains, likely reflecting that the ExoN domain must be present to enable N7-MTase activity. We further identify multi-functional host protein Tollip as an Nsp14 interactor. Interaction requires the phosphoinositide-binding C2 domain of Tollip and sequences C-terminal to the C2 domain. Full-length Tollip or regions encompassing the Nsp14 interaction domain are sufficient to counteract both Nsp14-mediated and Nsp14-independent activation of NF-κB. Knockdown of Tollip partially reverses IFNAR1 and IFNGR1 downregulation in SARS-CoV-2-infected cells, suggesting the relevance of Nsp14-Tollip interaction for Nsp14 innate immune evasion functions.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structural protein 14 (Nsp14) both activates NF-κB, which promotes virus replication and inflammation, and downregulates interferon alpha/beta receptor 1 (IFNAR1), which can render infected cells resistant to the antiviral effects of IFN-α/β. Our study demonstrates that Nsp14 also activates MAPK signaling and downregulates IFN-γ receptor 1 (IFNGR1), causing broader impacts than previously recognized. Data from a panel of Nsp14 mutants suggest that a common underlying effect of Nsp14 may be responsible for its multiple innate immune activities. We further describe a novel interaction between Nsp14 and Tollip, a selective autophagy receptor. We show that Tollip expression downregulates Nsp14 activation of NF-κB and that Tollip knockdown reverses IFNAR1 and IFNGR1 downregulation in SARS-CoV-2 infection, suggesting that Tollip functions as a regulator of Nsp14 innate immune modulation.