Willy J. M. Spaan

Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage.

Abstract The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.

Coronaviruses : structure and genome expression

Introduction. Progress in coronavirology is illustrated by the number of workshops convened and reviews written. International meetings have been held in Germany (1980), the Netherlands (1983) and the U.S.A. (1986), and the Fourth Coronavirus Symposium will be organized by one of us (D.C.) in Cambridge, U.K. in July 1989. In addition, reviews have appeared which highlighted particularly interesting characteristics of the family, e.g. the replication strategy (Lai, 1986) and the glycoproteins (Sturman & Holmes, 1985). As the last general accounts were published some 5 years ago (Siddell et al., 1983; Sturman & Holmes, 1983) an update is timely. The present article is based on the large amount of sequence data accumulated in these years and focuses on the viral nucleic acids and proteins and their function. Coronaviruses cause infections in man, other mammals and birds. Most experimental data have been obtained from studies of mouse hepatitis virus (MHV) and infectious bronchitis virus of chickens (IBV).

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.

Severe acute respiratory syndrome coronavirus phylogeny: toward consensus.

Since the identification of a new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) as the causative agent of the SARS epidemic in the winter of 2002-2003, the origin of the novel agent has remained a hotly debated topic. Which virus was the immediate ancestor of SARS-CoV, and what are the relationships between SARS-CoV and other previously described coronaviruses? Correct answers to these two questions are vital, as substantiated below, for designing strategies to detect, contain, and combat new outbreaks and for dissecting the fundamentals of the SARS-CoV life cycle. Major efforts have been invested in a thus far unsuccessful search for a natural SARS-CoV reservoir. In the meantime, and more outside the spotlight, SARS-CoV genome sequences have been used to define the phylogenetic position of SARSCoV among coronaviruses. These studies have resulted in a lot of controversy whose intricacies may not be very clear to outsiders. Our purpose is to clarify the situation from an insider’s point of view. Originally, coronaviruses were classified on the basis of antigenic cross-reactivity, and in this manner three antigenic groups (1 to 3) were recognized (14). When coronavirus genome sequences began to accumulate, the same groups were evident from phylogenetic analyses of the four structural proteins, N, M, E, and S (19), and of different regions of the giant replicase (3, 22). Group boundaries were also supported by the diversity of small open reading frames (ORFs) encoding accessory proteins, which are dispersed among the structural protein genes in the 3 -proximal region of the genome (Fig. 1). In the middle of the nineties, a first discord between the antigenicity-based and phylogenetic classifications emerged upon the characterization of the coronavirus porcine epidemic diarrhea virus (PEDV) and human coronavirus 229E (HCoV229E), one of the common cold viruses. These viruses proved not to have antigenic cross-reactivity with members of the established groups (18), yet on the basis of sequence comparisons it was concluded that they segregate into group 1, although they are somewhat separated from porcine transmissible gastroenteritis virus and closely related viruses (subgroup 1b and subgroup 1a, respectively, in Fig. 2) (9). The PEDV and HCoV-229E genomes also share an ORF specific for group 1 in the 3 -proximal region of their genome. The Coronavirus Study Group of the International Committee on Taxonomy of Viruses recognized these viruses as members of group 1 rather than declaring them prototypes of new groups (6). This decision effectively converted the original antigenic groups—which were based essentially on some properties of one or a few viral proteins—into a genetic one based on full-length genome sequences, but this change was never acknowledged explicitly. Consequently, no guidelines were established with respect to handling future disagreements between the classifications based on antigenicity, genome organization, and phylogeny should these arise from the properties of newly identified coronaviruses, and SARS-CoV proved to be quite a classification challenge. Initial phylogenetic analyses suggested that the novel virus did not cluster with any of the three established coronavirus groups. Accordingly, SARS-CoV also has a unique pattern of small ORFs in the 3 -proximal region of its genome and a unique internal organization of its nonstructural protein 3 (nsp3) replicase subunit, which includes a sizable novel domain (SARS-CoV unique domain SUD) and only one papain-like protease (PL2pro) rather than the two copies commonly found in other coronaviruses (Fig. 1). Although a thorough assessment of the antigenic cross-reactivity of SARS-CoV with other coronaviruses is yet to be published, a proposal to recognize SARS-CoV as a representative of a new, fourth group of coronaviruses seemed most logical (15, 17). If SARS-CoV indeed represents a new group, then when, relative to other groups, could this lineage have emerged? Several scenarios are theoretically plausible, and one of the most extreme ones, which seems compatible with the unique characteristics of SARS-CoV, places the origin of this lineage next to the ancestor of the other coronaviruses (Fig. 2A). To rigorously infer the origin of SARS-CoV, we conducted a special analysis of the replicase ORF1b region (Fig. 1), the mostconserved part of the coronavirus genome, which accounts for 20% of its size (20). In this analysis, the equine torovirus—a distant relative of coronaviruses belonging to the genus Torovirus of the same Coronaviridae family—was used as an outgroup to infer the direction of coronavirus evolution. Surprisingly, our fully resolved tree demonstrated that the SARS-CoV lineage is an early split-off from the group 2 branch and that the split-off occurred relatively late in coronavirus evolution, after the two bifurcations that gave rise to the three previously established groups (Fig. 2B). This topology is unlikely to be skewed, as it was obtained by using different criteria * Corresponding author. Mailing address: Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Albinusdreef 2, E4-P, Room L04-036, 2333 ZA Leiden, The Netherlands. Phone: 31-71-526-1652. Fax: 31-71-526-6761. E-mail: a.e.gorbalenya@lumc.nl.

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets.

Summary SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3·3 logs (95% Cl 2·6–4·0 logs; p<0·001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0·013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection.

ALMOST THE ENTIRE 5' NON-TRANSLATED REGION OF HEPATITIS C VIRUS IS REQUIRED FOR CAP-INDEPENDENT TRANSLATION

To investigate which hairpin structures within the 5′ untranslated region of hepatitis C virus (HCV) are necessary for cap‐independent translation, mutants were constructed that lack one or more hairpin structures. Here we demonstrate, by constructing precisely defined hairpin deletion mutants, that with the exception of the most 5′ located hairpin structure, which on deletion shows an increase on translation, each of the predicted hairpins is found to be essential for cap‐independent translation. In addition, we demonstrate that HCV 5′UTR driven translation is stimulated by poliovirus 2Apro co‐expression.

Sequence of mouse hepatitis virus A59 mRNA 2: Indications for RNA recombination between coronaviruses and influenza C virus

Abstract The nucleotide sequence of the unique region of coronavirus MHV-A59 mRNA 2 has been determined. Two open reading frames (ORF) are predicted: ORF1 potentially encodes a protein of 261 amino acids; its amino acid sequence contains elements which indicate nucleotide binding properties. ORF2 predicts a 413 amino acids protein; it lacks a translation initiation codon and is therefore probably a pseudogene. The amino acid sequence of ORF2 shares 30% homology with the HA1 hemagglutinin sequence of influenza C virus. A short stretch of nucleotides immediately upstream of ORF2 shares 83% homology with the MHC class I nucleotide sequences. We discuss the possibilitythat both similarities are the result of recombinations and present a model for the acquisition and the subsequent inactivation of ORF2; the model applies also to MHV-A59-related coronaviruses in which we expect ORF2 to be still functional.

Evidence for a coiled-coil structure in the spike proteins of coronaviruses

Abstract The amino acid sequences of the spike proteins from three distantly related coronaviruses have been deduced from cDNA sequences. In the C-terminal half, an homology of about 30% was found, while there was no detectable sequence conservation in the N-terminal regions. Hydrophobic “heptad” repeat patterns indicated the presence of two α-helices with predicted lengths of 100 and 50 Å, respectively. It is suggested that, in the spike oligomer. these α-helices form a complex coiled-coil, resembling the supersecondary structures in two other elongated membrane proteins, the haemagglutinin of influenza virus and the variable surface glycoprotein of trypanosomes.

Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site

Abstract The nucleotide sequence of the peplomer (E2) gene of MHV-A59 was determined from a set of overlapping cDNA clones. The E2 gene encodes a protein of 1324 amino acids including a hydrophobic signal peptide. A second large hydrophobic domain is found near the COOH terminus and probably represents the membrane anchor. Twenty glycosylation sites are predicted. Cleavage of the E2 protein results in two different 90K species, 90A and 90B (L. S. Sturman, C. S. Ricard, and K. V. Holmes (1985) J. Virol. 56, 904–911), and activates cell fusion. Protein sequencing of the trypsin-generated N-terminus revealed the position of the cleavage site. 90A and 90B could be identified as the C-terminal and the N-terminal parts, respectively. Amino acid sequence comparison of the A59 and 1HM E2 proteins showed extensive homology and revealed a stretch of 89 amino acids in the 90B region of the A59 E2 protein that is absent in JHM.

The Production of Recombinant Infectious DI-Particles of a Murine Coronavirus in the Absence of Helper Virus

Abstract We have studied the production and release of infectious DI-particles in vaccinia-T7-polymerase recombinant virus-infected L cells that were transfected with five different plasmids expressing the synthetic DI RNA MIDI-HD and the four structural proteins (M, N, S, and E) of the murine coronavirus MHV-A59. The DI cDNA contains the hepatitis delta ribozyme sequences to generate in the transfected cells a defined 3′ end. In EM studies of transfected cells virus-like particles (VLP) were observed in vesicles. Release of the particles into the medium was studied by immunoprecipitations of proteins released into the culture supernatant. Particle release was independent of S or N, but required M and E. Coexpression of E and M was sufficient for particle release. Coexpression of the structural proteins and the MIDI-HD RNA resulted in the production and release of infectious DI-particles. Infectivity of the DI-particles was determined by adding helper virus MHV-A59 to the medium containing the VLPs and using this mixture to infect new L cells. Intracellular RNA of several subsequent undiluted passages was isolated to detect the MIDI-HD RNA. Passage of the MIDI-HD RNA was dependent on the expression of the structural proteins of MHV-A59 in the transfected cells. In the absence of either E or M, MIDI-HD RNA could not be passaged to fresh L cells. We have thus developed a system in which we can produce coronavirus-like particles and an assay to test their infectivity.