Interplay between coronavirus, a cytoplasmic RNA virus, and nonsense-mediated mRNA decay pathway

Masami Wada,Kumari G Lokugamage,Keisuke Nakagawa,Krishna Narayanan,Shinji Makino

doi:10.1073/pnas.1811675115

Abstract

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals. Very little is known about the host pathways that regulate the stability of CoV mRNAs, which carry some unusual features. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features and targets them for degradation. Although CoV mRNAs are of cytoplasmic origin, the presence of several NMD-inducing features (including multiple ORFs with internal termination codons that create a long 3' untranslated region) in CoV mRNAs led us to explore the interplay between the NMD pathway and CoVs. Our study using murine hepatitis virus as a model CoV showed that CoV mRNAs are recognized by the NMD pathway as a substrate, resulting in their degradation. Furthermore, CoV replication induced the inhibition of the NMD pathway, and N protein (a viral structural protein) had an NMD inhibitory function that protected viral mRNAs from rapid decay. Our data further suggest that the NMD pathway interferes with optimal viral replication by degrading viral mRNAs early in infection, before sufficient accumulation of N protein. Our study presents clear evidence for the biological importance of the NMD pathway in controlling the stability of mRNAs and the efficiency of replication of a cytoplasmic RNA virus.

Highlights

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals
To test whether the cytoplasmically generated rLucRNA3 transcripts can serve as a substrate of the Nonsense-mediated decay (NMD) pathway, we examined the stability of rLucRNA3 transcripts in control siRNA-treated cells (NMD-competent) or in cells depleted of UPF1 or UPF2 (NMD-deficient)
To gain further evidence that newly synthesized mouse hepatitis virus (MHV) mRNA 1 is susceptible to NMD early in infection, we examined the effect of NMD inhibition on the stability of mRNA 1 in cells treated with E64d, a specific, irreversible inhibitor of cysteine proteinases that inhibits the processing of viral gene 1 polyprotein, resulting in the inhibition of CoV RNA synthesis [38]

Summary

Introduction

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, are enveloped RNA viruses that carry a large positive-sense single-stranded RNA genome and cause a variety of diseases in humans and domestic animals. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features and targets them for degradation. Our study presents clear evidence for the biological importance of the NMD pathway in controlling the stability of mRNAs and the efficiency of replication of a cytoplasmic RNA virus. RNA), several subgenomic mRNAs are synthesized in infected cells (SI Appendix, Fig. S1) These subgenomic mRNAs encode viral structural proteins and accessory proteins, the latter of which are not essential for virus replication in cell culture but play a role in viral pathogenicity [8, 9]. Nonsense-mediated decay (NMD) is a eukaryotic RNA surveillance pathway that detects mRNAs harboring aberrant features [e.g., premature termination codon (PTC)], and targets them for degradation [10]. Studies that lead to a comprehensive understanding of CoV gene expression strategies and host interactions will provide the necessary knowledge required for the development of new and effective measures to control CoV replication

Methods

Results

Conclusion