2006 [Advances in Virus Research] Volume 66 __ The Molecular Biology of Coronaviruses

THE MOLECULAR BIOLOGY OF CORONAVIRUSES Paul S. Masters Wadsworth Center, New York State Department of Health Albany, New York 12201 I. Introduction II. Taxonomy III. Virion Morphology, Structural Proteins, and Accessory Proteins A. Virus and Nucleocapsid B. Spike Protein (S) C. Membrane Protein (M) D. Envelope Protein (E) E. Nucleocapsid Protein (N) F. Genome G. Accessory Proteins IV. Viral Replication Cycle and Virion Assembly A. Receptors and Entry B. Virion Assembly Interactions C. Genome Packaging V. RNA Synthesis A. Replication and Transcription B. RNA Recombination C. Replicase Complex VI. Genetics and Reverse Genetics References ABSTRACT Coronaviruses are large, enveloped RNA viruses of both medical and veterinary importance. Interest in this viral family has intensified in the past few years as a result of the identification of a newly emerged coronavirus as the causative agent of severe acute respiratory syn- drome (SARS). At the molecular level, coronaviruses employ a variety of unusual strategies to accomplish a complex program of gene expres- sion. Coronavirus replication entails ribosome frameshifting during genome translation, the synthesis of both genomic and multiple sub- genomic RNA species, and the assembly of progeny virions by a path- way that is unique among enveloped RNA viruses. Progress in the investigation of these processes has been enhanced by the development of reverse genetic systems, an advance that was heretofore obstructed 193 0065-3527/06 $35.00 DOI: 10.1016/S0065-3527(06)66005-3 ADVANCES IN VIRUS RESEARCH, VOL 66 by the enormous size of the coronavirus genome. This review sum- marizes both classical and contemporary discoveries in the study of the molecular biology of these infectious agents, with particular emphasis on the nature and recognition of viral receptors, viral RNA synthesis, and the molecular interactions governing virion assembly. I. INTRODUCTION Coronaviruses are a family of enveloped RNA viruses that are distributed widely among mammals and birds, causing principally respiratory or enteric diseases but in some cases neurologic illness or hepatitis (Lai and Holmes, 2001). Individual coronaviruses usually infect their hosts in a species-specific manner, and infections can be acute or persistent. Infections are transmitted mainly via respiratory and fecal-oral routes. The most distinctive feature of this viral family is genome size: coronaviruses have the largest genomes among all RNA viruses, including those RNA viruses with segmented genomes. This expansive coding capacity seems to both provide and necessitate a wealth of gene-expression strategies, most of which are incompletely understood. Two prior reviews with the same title as this one have appeared in the Advances in Virus Research series (Lai and Cavanagh, 1997; Sturman and Holmes, 1983). The earlier of the two noted that the recognition of coronaviruses as a separate virus family occurred in the 1960s, in the wake of the discovery of several new human respira- tory pathogens, certain of which, it was realized, appeared highly similar to the previously described avian infectious bronchitis virus (IBV) and mouse hepatitis virus (MHV) (Almeida and Tyrrell, 1967). These latter viruses had a characteristic morphology in negative-stained electron microscopy, marked by a “fringe” of surface structures described as “spikes” (Berry et al., 1964) or “club-like” projections (Becker et al., 1967). Such structures were less densely distributed and differently shaped than those of the myxoviruses. To some, the fringe resembled the solar corona, giving rise to the name that was ultimately assigned to the group (Almeida et al., 1968). Almost four decades later, recognition of the same characteristic virion morphology alerted the world to the emer- gence of another new human respiratory pathogen: the coronavirus responsible for the devastating outbreak of severe acute respiratory syndrome (SARS) in 2002–2003 (Ksiazek et al., 2003; Peiris et al., 2003). The sudden appearance of SARS has stimulated a burst of new research to understand the basic replication mechanisms of members of 194 PAUL S. MASTERS this family of viral agents, as a means toward their control and prophy- laxis. Thus, the time is right to again assess the state of our collective knowledge about the molecular biology of coronaviruses. Owing to limitations imposed by both space and the expertise of the author, “molecular biology” will be considered here in the more narrow sense, that is, the molecular details of the cellular replication of coro- naviruses. No attempt will be made to address matters of pathogenesis, viral immunology, or epidemiology. For greater depth and differences of emphasis in particular areas, as well as for historical perspectives, the reader is referred to the two excellent predecessors of this review (Lai and Cavanagh, 1997; Sturman and Holmes, 1983) and also to volumes edited by Siddell (1995) and Enjuanes (2005). II. TAXONOMY Coronaviruses are currently classified as one of the two genera in the family Coronaviridae (Enjuanes et al., 2000b). However, it is likely that the coronaviruses, as well as the other genus within the Coronaviridae, the toroviruses (Snijder and Horzinek, 1993), will each be accorded the taxonomic status of family in the near future (Gonza´lez et al., 2003). Therefore, throughout this review, the coronaviruses are referred to as a family. Both the coronaviruses and the toroviruses, in addition to two other families, the Arteriviridae (Snijder and Meulenberg, 1998) and the Roniviridae (Cowley et al., 2000; Dhar et al., 2004), have been grouped together in the order Nidovirales. This higher level of organi- zation recognizes a relatedness among these families that sets them apart from other nonsegmented positive-strand RNA viruses. The most salient features that all nidoviruses have in common are: gene expres- sion through transcription of a set of multiple 30-nested subgenomic RNAs; expression of the replicase polyprotein via ribosomal frameshift- ing; unique enzymatic activities among the replicase protein products; a virion membrane envelope; and a multispanning integral membrane protein in the virion. The first of these qualities provides the name for the order, which derives from the Latin nido for nest (Enjuanes et al., 2000a). In contrast to their commonalities, however, nidovirus families differ from one another in distinct ways, most conspicuously in the numbers, types, and sizes of the structural proteins in their virions and in the morphologies of their nucleocapsids. A more detailed comparison of characteristics of these virus families has been given by Enjuanes et al. (2000b) and Lai and Cavanagh (1997). THE MOLECULAR BIOLOGY OF CORONAVIRUSES 195 Members of the coronavirus family have been sorted into three groups (Table I), which, it has been proposed, are sufficiently divergent to merit the taxonomic status of genera (Gonza´lez et al., 2003). Classi- fication into groups was originally based on antigenic relationships. However, such a criterion reflects the properties of a limited subset of viral proteins, and cases have arisen where clearly related viruses in group 1 were found not to be serologically cross-reactive (Sanchez et al., 1990). Consequently, sequence comparisons of entire viral gen- omes (or of as much genomic sequence as is available) have come to be the basis for group classification (Gorbalenya et al., 2004). Almost all group 1 and group 2 viruses have mammalian hosts, with human coronaviruses falling into each of these groups. Viruses of group 3, by contrast, have been isolated solely from avian hosts. Most of the cor- onaviruses in Table I have been studied for decades, and, by the turn of the century, the scope of the family seemed to be fairly well-defined. Accordingly, it came as quite a shock, in 2003, when the causative agent of SARS was found to be a coronavirus (SARS-CoV). Equally astonishing have been the outcomes of renewed efforts, following the SARS epidemic, to detect previously unknown viruses; these investi- gations have led to the discovery of two more human respiratory coronaviruses, HCoV-NL63 (van der Hoek et al., 2004) and HCoV- HKU1 (Woo et al., 2005). Three distinct bat coronaviruses have also been isolated: two are members of group 1, and the third, in group 2, is a likely precursor of the human SARS-CoV (Lau et al., 2005; Li et al., 2005c; Poon et al., 2005). In addition, new IBV-like viruses have been found that infect geese, pigeons, and ducks (Jonassen et al., 2005). In almost all cases, the assignment of a coronavirus species to a given group has been unequivocal. Exceptionally, the classification of SARS- CoV has provoked considerable controversy. The original, unrooted, phylogenetic characterizations of the SARS-CoV genome sequence pos- ited this virus to be roughly equidistant from each of the three previ- ously established groups. It was thus proposed to be the first recognized member of a fourth group of coronaviruses (Marra et al., 2003; Rota et al., 2003). However, a subsequently constructed phylogeny based on gene 1b, which contains the viral RNA-dependent RNA polymerase and which was rooted in the toroviruses as an outgroup, concluded that SARS-CoV is most closely related to the group 2 coronaviruses (Snijder et al., 2003). In the same vein, it was noted that regions of gene 1a of SARS-CoV contain domains that are unique to the group 2 corona- viruses (Gorbalenya et al., 2004). Other analyses of a subset of structur- al gene sequences (Eickmann et al., 2003) and of RNA secondary structures in the 30 untranslated region (30 UTR) of the genome (Goebel 196 PAUL S. MASTERS TABLE I CORONAVIRUS SPECIES AND GROUPS Group Designation Species Host GenBank accession number* 1 TGEV Transmissible gastroenteritis virus Pig AJ271965 [g] PRCoV Porcine respiratory coronavirus Pig Z24675 [p] FIPV Feline infectious peritonitis virus Cat AY994055 [g] FCoV Feline enteric coronavirus Cat Y13921 [p] CCoV Canine coronavirus Dog D13096 [p] HCoV-229E Human coronavirus strain 229E Human AF304460 [g] PEDV Porcine epidemic diarrhea virus Pig AF353511 [g] HCoV-NL63 Human coronavirus strain NL63 Human AY567487 [g] Bat-CoV-61 Bat coronavirus strain 61 Bat AY864196 [p] Bat-CoV-HKU2 Bat coronavirus strain HKU2 Bat AY594268 [p] 2 MHV Mouse hepatitis virus Mouse AY700211 [g] BCoV Bovine coronavirus Cow U00735 [g] RCoV Rat coronavirus Rat AF088984 [p] SDAV Sialodacryoadenitis virus Rat AF207551 [p] HCoV-OC43 Human coronavirus strain OC43 Human AY903460 [g] HEV Hemagglutinating encephalomyelitis virus Pig AF481863 [p] PCoV† Puffinosis coronavirus Puffin AJ544718 [p] ECoV Equine coronavirus Horse AY316300 [p] CRCoV Canine respiratory coronavirus Dog CQ772298 [p] SARS-CoV SARS coronavirus Human AY278741 [g] HCoV-HKU1 Human coronavirus strain HKU1 Human AY597011 [g] Bat-SARS-CoV Bat SARS coronavirus Bat DQ022305 [g] (continues) THE MOLECULAR BIOLOGY OF CORONAVIRUSES 197 et al., 2004b) also supported a group 2 assignment. By contrast, some authors have argued, based on bioinformatics methods, that the ancestor of SARS-CoV was derived from multiple recombination events among progenitors from all three groups (Rest and Mindell, 2003; Stanhope et al., 2004; Stavrinides and Guttman, 2004). While these latter studies assume that historically there has been limitless opportunity for inter- group recombination, there is no well-documented example of recombi- nation between extant coronaviruses of different groups. Moreover, it is not clear that intergroup recombination is even possible, owing to repli- cative incompatibilities among the three coronavirus groups (Goebel et al., 2004b). Therefore, although SARS-CoV does indeed have unique features, the currently available evidence best supports the conclusion that it is more closely allied with the group 2 coronaviruses and that it has not sufficiently diverged to constitute a fourth group (Gorbalenya et al., 2004). III. VIRION MORPHOLOGY, STRUCTURAL PROTEINS, AND ACCESSORY PROTEINS A. Virus and Nucleocapsid Coronaviruses are roughly spherical and moderately pleiomorphic (Fig. 1). Virions have typically been reported to have average dia- meters of 80–120 nm, but extreme sizes as small as 50 nm and as large TABLE I (continued) Group Designation Species Host GenBank accession number* 3 IBV Infectious bronchitis virus Chicken AJ311317 [g] TCoV Turkey coronavirus Turkey AY342357 [p] PhCoV Pheasant coronavirus Pheasant AJ618988 [p] GCoV Goose coronavirus Goose AJ871017 [p] PCoV† Pigeon coronavirus Pigeon AJ871022 [p] DCoV Duck coronavirus Mallard AJ871024 [p] * One representative GenBank accession number is given for each species. When available, a complete genomic sequence (denoted [g]) is provided; otherwise, the largest available partial sequence (denoted [p]) is given. † Unique designations have not yet been formulated for these two viruses. 198 PAUL S. MASTERS as 200 nm are occasionally given in the older literature (Oshiro, 1973; McIntosh, 1974). The surface spikes or peplomers of these viruses, vari- ously described as club-like, pear-shaped, or petal-shaped, project some 17–20 nm from the virion surface (McIntosh, 1974), having a thin base that swells to a width of about 10 nm at the distal extremity (Sugiyama and Amano, 1981). For some coronaviruses a second set of projections, 5–10-nm long, forms an undergrowth beneath the major spikes (Guy et al., 2000; Patel et al., 1982; Sugiyama and Amano, 1981). These shorter structures are now known to be the hemagglutinin-esterase (HE) protein that is found in a subset of group 2 coronaviruses (Section III.G). At least some of the heterogeneity in coronavirus particle morphology can be attributed to the distorting effects of negative-staining proce- dures. Freeze-dried (Roseto et al., 1982) and cryo-electron microscopic (Risco et al., 1996) preparations of BCoV and TGEV, respectively, showed much more homogeneous populations of virions, with diameters 10–30 nm greater than virions in comparable samples prepared by negative staining. Extraordinary three-dimensional images have been obtained for SARS-CoV virions emerging from infected Vero cells (Ng et al., 2004). These scanning electron micrographs and atomic force micrographs reveal knobby, rosette-like viral particles resembling tiny cauliflowers. It will be exciting to see future applications of advanced imaging techniques to the study of coronavirus structure. The internal component of the coronavirus virion is obscure in elec- tron micrographs of whole virions. In negative-stained images the FIG 1. Schematic of the coronavirus virion, with the minimal set of structural proteins. THE MOLECULAR BIOLOGY OF CORONAVIRUSES 199 core appears as an indistinct mass with a densely staining center, giving the virion a “punched-in” spherical appearance. Imaging of virions that have burst spontaneously, expelling their contents, or that have been treated with nonionic detergents has allowed visuali- zation of the coronavirus core. Such analyses led to the attribution of another distinguishing characteristic to the coronavirus family: that its members possess helically symmetric nucleocapsids. Such nucleo- capsid symmetry is the rule for negative-strand RNA viruses, but almost all positive-strand RNA animal viruses have icosahedral ribo- nucleoprotein capsids. However, although it is fairly well accepted that coronaviruses have helical nucleocapsids, there are surprisingly few published data that bear on this issue. Additionally, the reported results vary considerably with both the viral species and the method of prepara- tion. The earliest study of nucleocapsids from spontaneously disrupted HCoV-229E virions found tangled, threadlike structures 8–9 nm in diam- eter; these were unraveled or clustered to various degrees and, in rare cases, retained some of the shape of the parent virion (Kennedy and Johnson-Lussenburg, 1975/76). A subsequent analysis of spontaneously disrupted virions of HCoV-229E and MHV observed more clearly helical nucleocapsids, with diameters of 14–16 nm and hollow cores of 3–4 nm (Macnaughton et al., 1978). The most highly resolved images of any coronavirus nucleocapsid were obtained with NP-40-disrupted HCoV- 229E virions (Caul et al., 1979). These preparations showed filamentous structures 9–11 or 11–13 nm in diameter, depending on the method of staining, with a 3–4-nm central canal. The coronavirus nucleocapsid was noted to be thinner in cross-section than those of paramyxoviruses and also to lack the sharply segmented “herringbone” appearance character- istic of paramyxovirus nucleocapsids. By contrast, in early studies, IBV and TGEV nucleocapsids were refractory to the techniques that had been successful with other viruses. Visualization of IBV nucleocapsids, which seemed to be very sensitive to degradation (Macnaughton et al., 1978), was finally achieved by electron microscopy of viral samples prepared by carbon-platinum shadowing (Davies et al., 1981). This revealed linear strands, some as long as 6–7 mm, which were only 1.5-nm thick, suggest- ing that they represented unwound helices. TGEV, on the other hand, was found to be more resistant to nonionic detergents. Treatment of virions of this species with NP-40 resulted in spherical subviral particles with no threadlike substructure visible (Garwes et al., 1976). The TGEV core was later seen as a spherically symmetric, possibly icosahedral, superstructure that only dissociated further into a helical nucleocapsid following Triton X-100 treatment of virions (Risco et al., 1996). Such a collection of incomplete and often discrepant results makes it clear that 200 PAUL S. MASTERS much further examination of the internal structure of coronavirus vir- ions is warranted. It would substantially aid our understanding of coro- navirus structure and assembly if we had available a detailed description of nucleocapsid shape, length, diameter, helical repeat distance, and protein:RNA stoichiometry. B. Spike Protein (S) There are three protein components of the viral envelope (Fig. 1). The most prominent of these is the S glycoprotein (formerly called E2) (Cavanagh, 1995), which mediates receptor attachment and viral and host cell membrane fusion (Collins et al., 1982). The S protein is a very large, N-exo, C-endo transmembrane protein that assembles into tri- mers ( Delmas and Laude, 1990; S ong et al., 2004) to form the dist inc- tive surface spikes of coronaviruses (Fig. 2). S protein is inserted into the endoplasmic reticulum (ER) via a cleaved, amino-terminal signal peptide (Cavanagh et al., 1986b). The ectodomain makes up most of the molecule, with only a small carboxy-terminal segment (of 71 or fewer of the total 1162–1452 residues) constituting the transmembrane domain and endodomain. Monomers of S protein, prior to glycosylation, are 128–160 kDa, but molecular masses of the glycosylated forms of FIG 2. The spike (S) protein. At the right is a linear map of the protein, denoting the amino-terminal S1 and the carboxy-terminal S2 portions of the molecule. The arrow- head marks the site of cleavage for those S proteins that become cleaved by cellular protease(s). The signal peptide and regions of mapped receptor-binding domains (RBDs) are shown in S1. The heptad repeat regions (HR1 and HR2), putative fusion peptide (F), transmembrane domain, and endodomain are indicated in S2. At the left is a model for the S protein trimer. THE MOLECULAR BIOLOGY OF CORONAVIRUSES 201 full-length monomers fall in the range of 150–200 kDa. The S molecule is thus highly glycosylated, and this modification is exclusively N-linked (Holmes et al., 1981; Rottier et al., 1981). S protein ecto- domains have from 19 to 39 potential consensus glycosylation sites, but a comprehensive mapping of actual glycosylation has not yet been reported for any coronavirus. A mass spectrometric analysis of the SARS-CoV S protein has shown that at least 12 of the 23 candidate sites are glycosylated in this molecule (Krokhin et al., 2003). For the TGEV S protein, it has been demonstrated that the early steps of glycosylation occur cotranslationally, but that terminal glycosylation is preceded by trimerization, which can be rate-limiting in S protein maturation (Delmas and Laude, 1990). In addition, glycosylation of TGEV S may assist monomer folding, given that tunicamycin inhibi- tion of high-mannose transfer was found to also block trimerization. The S protein ectodomain has between 30 and 50 cysteine residues, and within each coronavirus group the positions of cysteines are well conserved (Abraham et al., 1990; Eickmann et al., 2003). However, as with glycosylation, a comprehensive mapping of disulfide linkages has not yet been achieved for any coronavirus S protein. In most group 2 and all group 3 coronaviruses, the S protein is cleaved by a trypsin-like host protease into two polypeptides, S1 and S2, of roughly equal sizes. Even for uncleaved S proteins, that is, those of the group 1 coronaviruses and SARS-CoV, the designations S1 and S2 are used for the amino-terminal and carboxy-terminal halves of the S protein, respectively. Peptide sequencing has shown that cleavage occurs following the last residue in a highly basic motif: RRFRR in IBV S protein (Cavanagh et al., 1986b), RRAHR in MHV strain A59 S protein (Luytjes et al., 1987), and KRRSRR in BCoV S protein (Abraham et al., 1990). Similar cleavage sites are predicted from the sequences of other group 2 S proteins, except that of SARS-CoV. It has been noted that the S protein of MHV strain JHM has a cleavage motif (RRARR) more basic than that found in MHV strain A59 (RRAHR). An expression study has shown that this difference accounts for the al- most total extent of cleavage of the JHM S protein that is seen in cell lines in which the A59 S protein undergoes only partial cleavage (Bos et al., 1995). The S1 domain is the most divergent region of the molecule, both across and within the three coronavirus groups. Even among strains and isolates of a single coronavirus species, the sequence of S1 can vary extensively (Gallagher et al., 1990; Parker et al., 1989; Wang et al., 1994). By contrast, the most conserved part of the molecule across the three coronavirus groups is a region that encompasses the 202 PAUL S. MASTERS