NUDT2 initiates viral RNA degradation by removal of 5\u2032-phosphates

Beatrice T Laudenbach,Alexander Reim,Marco Binder,Andreas Pichlmair,Quirin Emslander,Karsten Krey,Katrin Manske,Andrea Kröger,Line Lykke Andersen,Sarah Mundigl,Christopher Dächert,Janos Ludwig,Pietro Scaturro,Markus Moser,Dirk Wohlleber

doi:10.1038/s41467-021-27239-y

Abstract

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5′-triphosphate (PPP-) group that impairs degradation by the canonical 5′-3′ degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5′-3′ exonuclease XRN1. NUDT2 removes 5′-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5′-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.

Highlights

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA
To establish a screening system that allows testing for 5′-to-3′ dependent RNA degradation in virus-infected cells, we first evaluated whether CRISPR/Cas9-mediated depletion of XRN1 affects the replication of PPP-RNA-generating vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP)
Automated live-cell microscopy of VSV-GFP infected cells showed a distinct increase of GFP signal in XRN1 targeted cells as compared to controls (Fig. 1b), indicating that VSV-GFP is restricted by a 5′to-3′ exonuclease dependent pathway

Summary

Introduction

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. The 5′ phosphates can be removed by the RNA pyrophosphohydrolase (RppH) to prepare RNA for degradation by the bacterial 5′-to-3′ endoribonuclease RNase E or 5′ exonucleases (e.g., RNase J11–13 (Fig. 1a)) Both mammalian DCP2 and bacterial RppH belong to the superfamily of Nudix hydrolases[14], a protein family that is highly diverse in terms of sequence, domain organization, and substrate specificity[15]. Since Nudix hydrolases process mammalian and bacterial mRNAs, we tested whether mammalian Nudix hydrolases may have dephosphorylation activity on PPP-RNA to initiate further exonucleolytic processing Understanding such viral RNA degradation mechanisms is essential to further study cellular antiviral principles that are in place to limit the spread and pathogenicity of infectious pathogens and their involvement in antiviral immunity

Methods

Results

Conclusion