The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling

Lilian Cristina Russo,Rebeka Tomasin,Isaac Araújo Matos,Antonio Carlos Manucci,Sven T Sowa,Katie Dale,Keith W Caldecott,Lari Lehtiö,Deborah Schechtman,Flavia C Meotti,Alexandre Bruni-Cardoso,Nicolas Carlos Hoch

doi:10.1016/j.jbc.2021.101041

Lilian Cristina Russo, Rebeka Tomasin + Show 10 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101041

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Abstract

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.

Highlights

Type I IFN signaling is initiated upon recognition of viral nucleic acids in the cytoplasm, leading to the production and secretion of type I IFNs, such as IFNα and IFNβ, by virusinfected cells, whereas type II IFN, or IFNγ, is secreted by immune cells [1, 4]
We propose that PARP9/DTX3Ldependent ADP-ribosylation of host proteins, which can be reversed by the SARS-CoV-2 nonstructural protein 3 (Nsp3) macrodomain, is a downstream effector of the IFN response
The cells used in this analysis were gated to ensure similar expression levels between WT and N40A mutant macrodomain, with no effect on the result (Fig. S2, F and G). These data indicate that the Nsp3 macrodomain, when ectopically expressed in human cells, hydrolyzes the ADP-ribosylation of host proteins induced by both type I or type II IFN signaling

Summary

Introduction

Type I IFN signaling is initiated upon recognition of viral nucleic acids in the cytoplasm, leading to the production and secretion of type I IFNs, such as IFNα and IFNβ, by virusinfected cells, whereas type II IFN, or IFNγ, is secreted by immune cells [1, 4]. We show that activation of Type I or Type II IFN signaling induces ADP-ribosylation of host proteins that can be reversed by ectopic expression of the SARS-CoV-2 Nsp3 macrodomain in human cells.

Results

Conclusion