Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus. Ribosome profiling is emerging as a powerful technique to monitor translation in living cells at sub-codon resolution. It has particular applicability to virology, with the capacity to identify viral mRNAs that are being translated during infection and to provide new insights into virus gene expression, regulation and host-virus interactions. In this work, we carried out the first ribosome profiling analysis of an RNA virus, using as a model system the murine coronavirus strain MHV-A59, a betacoronavirus in the same genus as the medically important SARS-CoV and MERS-CoV. Parallel ribosome profiling and RNA sequencing of infected-cell time points was performed during the course of MHV replication in mouse tissue culture cells and used to determine virus gene expression kinetics and the relative translational efficiencies of virus and host mRNAs. The sensitivity and precision of the approach permitted us to uncover several unanticipated features of coronavirus translation, giving insights into ribosomal frameshifting, ribosome pausing, and the utilisation of short, potentially regulatory, upstream open reading frames. We also identified some challenges associated with the technique that are of general relevance to the ribosome profiling technique and developed bioinformatic strategies to address these.

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.

Viral capsids are envisioned as vehicles to deliver the viral genome to the host cell. They are nonetheless dynamic protective shells, as they participate in numerous processes of the virus cycle such as assembly, genome packaging, binding to receptors, and uncoating among others. In so doing, they undergo large scale conformational changes. Capsid proteins with essential enzymatic activities are being described more frequently. Here we show that the precursor (pVP2) of the capsid protein VP2 of the infectious bursal disease virus (IBDV), an avian double-stranded RNA virus, has autoproteolytic activity. The pVP2 C-terminal region is first processed by the viral protease VP4. VP2 Asp-431, lying in a flexible loop preceding the C-terminal most alpha-helix, is responsible for the endopeptidase activity that cleaves the Ala-441-Phe-442 bond to generate the mature VP2 polypeptide. The D431N substitution abrogates the endopeptidase activity without introducing a significant conformational change, as deduced from the three-dimensional structure of the mutant protein at 3.1 A resolution. Combinations of VP2 polypeptides containing mutations affecting either the cleavage or the catalytic site revealed that pVP2 proteolytic processing is the result of a monomolecular cis-cleavage reaction. The D431N mutation does not affect the assembly of the VP2 trimers that constitute the capsid building block. Although VP2 D431N trimers are capable of assembling both pentamers and hexamers, expression of a polyprotein gene harboring the D431N mutation does not result in the assembly of IBDV virus-like particles. Reverse genetics analyses demonstrate that pVP2 self-processing is essential for the assembly of an infectious IBDV progeny.