Figure 1 Coronavirus genome organization and expression. (Upper panel) Schematic representation of the genome of mouse hepatitis virus (MHV) shown as an example. ORFs are represented by boxes, indicated by number (above) and encoded protein (acronyms below). Regions encoding key domains in replicase polyproteins pp1a and pp1ab are colour-coded with hydrophobic segments shown in dark grey. The 5′-leader sequence is depicted by a small red box. The arrow between ORF 1a and 1b represents the ribosomal frameshifting site. The poly(A) tail is indicated by “A(n)”. Red arrowheads indicate the locations of transcription-regulating sequences (TRSs). PL (green) papain-like proteinase 1 (PL1^pro); PL (red), papain-like proteinase 2 (PL2^pro); A, ADP-ribose-1″phosphatase (macrodomain); M^pro, 3C-like main protease; Pr, noncanonical RNA-dependent RNA polymerase, putative primase; RdRp, RNA-dependent RNA polymerase; Z, zinc-binding domain; Hel, helicase domain; Exo, 3′ to-5′ exoribonuclease domain; N7, guanine-N7-methyltransferase; U, nidoviral uridylate-specific endoribonuclease (NendoU); MT, ribose-2′-O-methyltransferase domain; HE, hemagglutinin-esterase; S, spike protein; E, envelope protein; M, membrane protein, N, nucleocapsid protein; I, internal ORF. (Lower panel) Processing of the replicase polyproteins and structural relationship between the genomic RNA and subgenomic mRNAs of coronaviruses. Arrows indicate cleavage sites for PL1^pro (green), PL2^pro (red) and M^pro (blue). The locations of the non-structural proteins (nsp’s) are indicated by their number (see also Table 2). mRNA species are numbered as by convention on the basis of their size, from large to small, with the genome designated as RNA1. For the sg mRNAs only ORF(s) that are translated are shown.

Figure 2 Phylogenetic relationships among the members of the subfamily Coronavirinae. A rooted neighbor-joining tree was generated from amino acid sequence alignments of RdRp and helicase domains with equine torovirus Berne as outgroup. The tree reveals four main monophyletic clusters corresponding to genera Alpha-, Beta- and Gammacoronavirus and an envisaged new genus (color-coded), and also shows the distinct betacor­onavirus lineages A through D.

Figure 3 Coronavirus virion morphology. (A,B) Negative staining (2% phosphotungstic acid) electron micrographs of murine coronavirus particles. Shown are (A) a virion of murine coronavirus laboratory strain A59 that lacks HE expression and (B) one of a recombinant MHV-59 virus in which HE expression was restored (B) (courtesy Jean Lepault, Laboratory of Molecular and Structural Virology, Gif-sur-Yvette Cedex, France). (C,D) Cryo-electron tomographs of mouse hepatitis virus. A virtual slice (7.5 nm thick) through a reconstructed MHV particle (left) with highlighted features superimposed (right). The envelope is colored in orange with conspicuous striations highlighted; the nucleocapsid region is colored in blue. Note low-density region (ca. 4 nm) between envelope and nucleocapsid (reprinted with permission from Barcéna et al. (2008) Proc. Natl Acad. Sci., U S A, 106, 582-587, © 2008 National Academy of Sciences, USA). (E) Schematic representation of a (lineage A) betacoronavirus virion.

Figure 4 Coronavirus mRNA synthesis: the discontinuous 3′-extension model. Minus-strand synthesis initiates at the 3′ end of the genome and proceeds until a TRS is copied (1). The nascent minus-strand RNA may then be transferred to the 5′ end of the genome (2). Base complementarity allows the minus-strand RNA to anneal to the leader TRS (3) after which RNA synthesis resumes and body (in blue) and leader sequences (in red) become fused (4). The chimeric sg minus-strand RNA in turn serves as a template for “continuous” synthesis of sg mRNAs (5).

Figure 5 Alphacoronavirus genome organization. Comparison of the 3′-terminal genomic regions downstream of ORF1b of alphacoronaviruses representative of the different species and subspecies. ORFs are depicted as coloured boxes and indicated by number (above) and encoded protein. ORFs for accessory proteins are named as by convention according to number (referring to the mRNA species from which they are expressed) and, in the case of multiple ORFs in one transcription unit, alphabetically. Conservation of genes is indicated by identical colouring. Accessory genes of different viruses that are located in the same genomic location but believed to encode non-related products are coloured differently. For the abbreviations of virus names, please see list of species in the genus Alphacoronavirus below. 1b, ORF1b; mp, alphacoronavirus-specific accessory membrane protein αmp; all other acronyms as in Figure 1.

Figure 6 Betacoronavirus genome organization. Comparison of the 3′-terminal genomic regions downstream of ORF1b of betacoronaviruses representative for the different species and subspecies. ORFs are depicted as boxes, color-coded and indicated by number and gene product as in Figure 5. For the abbreviations of virus names, please see the list of species in the genus Betacoronavirus.

Figure 7 Gammacoronavirus genome organization. Comparison of the 3′-terminal genomic regions downstream of ORF1b of infectious bronchitis virus (IBV; sp. Avian coronavirus) and Beluga whale coronavirus SW1 (BWCoV). ORFs are depicted as boxes, colour-coded and indicated by number and gene product as in Figures 5 and 6.

Figure 8 Virion morphology of equine torovirus Berne. (A) Negative staining electron micrograph of extracellular EToV particles (2% phosphotungstic acid). (B) Close-up of negatively-stained EToV virions. (Courtesy of Dolores Rodriguez Aguirre, Department of Molecular and Cell Biology, National Centre of Biotechnology, Madrid, Spain.) Note that in EToV strain Berne, the HE gene is inactivated and that virions consequently display only one type of spike, the peplomers comprised of the S protein. (C) Schematic representation of a torovirus virion. (D and E) Cross-sections of intracellular EToV virions showing the tubular nucleocapsid with central cavity and the viral envelope. The bar represents 100 nm.

Figure 9 Organization and expression of the torovirus genome. (Upper panel) Schematic representation of the equine torovirus genome. ORFs and other genome elements are indicated as in Figure 1. The 5′-leader sequence present in the genome and sg mRNA 2 is depicted by a small red box. Green and red arrowheads/boxes indicate the locations of the internal and the 5′-terminal putative terminator/promoter (TP) elements, respectively. Blue arrows indicate established M^pro cleavage sites. The location of the discontinuous transcription element (DTE) driving mRNA 2 synthesis is shown by a hairpin. PL, papain-like proteinase; C, torovirus-specific ORF1a-encoded cyclic nucleotide phosphodiesterase domain. All other acronyms as in Figure 1. (inset) Structure of the mRNA 2 discontinuous transcription element, showing the hairpin structure and downstream “homology region” with sequence identity to the 5′ end of the genome indicated by asterisks. A hairpin residue-pair displaying co-variation in BToV and PToV is highlighted in green. The site of mRNA 2 leader–body fusion is indicated by an arrowhead. The 5′-terminal genomic TP copy is highlighted by a red box. (Lower panel) Models for discontinuous (left) and non-discontinuous sg RNA synthesis (right) in toroviruses. The hairpin indicates the mRNA 2 DTE. Red boxes correspond to the 5′-terminal genomic TP copy and complementary sequences, blue boxes to the DTE homology region and the corresponding 5′ genomic acceptor sequence. The TP consensus sequence is shown and highlighted by a green box. Internal TPs and complementary sequences are shown in green. The models show (from top to bottom) synthesis of genome-templated minus-strand RNA (minus-strand RNAs indicated by a wiggly line), attenuation and 3′-discontinuous extension directed by the DTE element and premature termination directed by internal TPs, and subsequent mRNA synthesis from sg minus-strand templates. Details are described in the text.

Figure 10 Genome organization of torovirus genotypes reveals evidence for multiple interspecies RNA recombination events. The genes for the replicase polyproteins (1a, 1b) and for the structural proteins S, M, HE, and N are depicted as boxes. For PToV, HE coding sequences are shown in different colors to indicate that one of the genotypes exchanged part of its HE gene through homologous RNA recombination with an as yet unknown torovirus. For BToV, sequences acquired from PToV (in yellow) and from a hitherto unidentified torovirus donor (in green) are also indicated.

Figure 11 Virion morphology and morphogenesis of white bream virus. (A) Negative staining electron micrograph of extracellular WBV particles (2% phosphotungstic acid, pH 7.4) (courtesy Harald Granzow and Thomas C. Mettenleiter, Friedrich Loeffler Institut, Bundesforschungsinstitut für Tiergesundheit, Greifswald Insel Riems, Germany). (B) Schematic representation of the WBV virion. (C and D) Cross-sections of (C) intracytoplasmic nucleocapsids and (D) a virion, with the nucleocapsid seemingly organized by subunits arranged in helical symmetry. (E) Preformed WBV nucleocapsids in the cytoplasm arranged side by side at smooth membranes. All bars represent 100 nm. (C, D and E from Granzow et al. (2001). Identification and ultrastructural characterization of a novel virus from fish.

J. Gen. Virol., 82, 2849-2859; with permission.)

Figure 12 Organization and expression of the WBV genome. Schematic representation of the genome of white bream virus and structural relationship between the genomic RNA and subgenomic mRNAs. 5′ leader sequences are indicated by red boxes, TRSs by blue arrowheads and boxes. Other genome elements and acronyms as in Figure 1.