Stuart G. Siddell

Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015)

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

Ribose 2′- O -methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5

The 5′ cap structures of higher eukaryote mRNAs have ribose 2′-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2′-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2′-O-methylation of mRNA remains elusive. Here we show that 2′-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2′-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2′-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2′-O-methylation of mRNA suggests that RNA modifications such as 2′-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.

The biology of coronaviruses.

Introduction. The Coronaviridae is a monogeneric family comprising 11 viruses which infect vertebrates. Members of the group are responsible for diseases of clinical and economic importance, in particular respiratory and gastrointestinal disorders (Table 1). The group was originally recognized on the basis of a characteristic virion morphology (Tyrrell et al., 1968), but can now be defined by biological and molecular criteria. Various aspects of coronavirus biology have been dealt with in recent reviews (Robb & Bond, 1979; Siddell et al., 1982; Wege et al., 1982). Structure. Morphology. Coronavirions are pleomorphic, although generally spherical, 60 to 220 nm in diameter and bear widely spaced, club-shaped surface projections about 20 nm in length. Complete virions have a density in sucrose of about 1.18 g/ml. In thin sections the virion envelope may be visualized as inner and outer shells separated by a translucent space. In negatively stained preparations of avian infectious bronchitis virus (IBV) an inner tongue-shaped membrane is visible (Bingham & Almeida, 1977; Fig. 1).

A Contemporary View of Coronavirus Transcription

Coronaviruses are a family of enveloped, plus-stranded RNA viruses with helical nucleocapsids and extraordinarily large genomes. The hallmark of coronavirus transcription is the production of multiple subgenomic mRNAs that contain sequences corresponding to both ends of the genome. (Transcription is

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain.

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, Mpro, a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus Mpro is reported. The structure was refined to 1.96 Å resolution and revealed three dimers in the asymmetric unit. The mutual arrangement of the protomers in each of the dimers suggests that Mpro self‐processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin‐like fold that is connected by a 16 residue loop to an extra domain featuring a novel α‐helical fold. Molecular modelling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of Mpro substrates. Interactions involving the N‐terminus and the α‐helical domain stabilize the loop in the orientation required for trans‐cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.

Coronavirus mRNA synthesis involves fusion of non-contiguous sequences.

Positive‐stranded genomic RNA of coronavirus MHV and its six subgenomic mRNAs are synthesized in the cytoplasm of the host cell. The mRNAs are composed of leader and body sequences which are non‐contiguous on the genome and are fused together in the cytoplasm by a mechanism which appears to involve an unusual and specific ‘polymerase jumping’ event.

Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

ABSTRACT Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.

Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017)

This article lists the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2017.

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single β-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).