Evolutionary Dynamics and Epidemiology of Endemic and Emerging Coronaviruses in Humans, Domestic Animals, and Wildlife.

Diverse coronavirus (CoV) strains can infect both humans and animals and produce various diseases. CoVs have caused three epidemics and pandemics in the last two decades, and caused a severe impact on public health and the global economy. Therefore, it is of utmost importance to understand the emergence and evolution of endemic and emerging CoV diversity in humans and animals. For diverse bird species, the Infectious Bronchitis Virus is a significant one, whereas feline enteric and canine coronavirus, recombined to produce feline infectious peritonitis virus, infects wild cats. Bovine and canine CoVs have ancestral relationships, while porcine CoVs, especially SADS-CoV, can cross species barriers. Bats are considered as the natural host of diverse strains of alpha and beta coronaviruses. Though MERS-CoV is significant for both camels and humans, humans are nonetheless affected more severely. MERS-CoV cases have been reported mainly in the Arabic peninsula since 2012. To date, seven CoV strains have infected humans, all descended from animals. The severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) are presumed to be originated in Rhinolopoid bats that severely infect humans with spillover to multiple domestic and wild animals. Emerging alpha and delta variants of SARS-CoV-2 were detected in pets and wild animals. Still, the intermediate hosts and all susceptible animal species remain unknown. SARS-CoV-2 might not be the last CoV to cross the species barrier. Hence, we recommend developing a universal CoV vaccine for humans so that any future outbreak can be prevented effectively. Furthermore, a One Health approach coronavirus surveillance should be implemented at human-animal interfaces to detect novel coronaviruses before emerging to humans and to prevent future epidemics and pandemics.

Protein Nucleotidylylation in +ssRNA Viruses.

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5' end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

In Vitro Models for Studying Entry, Tissue Tropism, and Therapeutic Approaches of Highly Pathogenic Coronaviruses.

Coronaviruses (CoVs) are enveloped nonsegmented positive-sense RNA viruses belonging to the family Coronaviridae that contain the largest genome among RNA viruses. Their genome encodes 4 major structural proteins, and among them, the Spike (S) protein plays a crucial role in determining the viral tropism. It mediates viral attachment to the host cell, fusion to the membranes, and cell entry using cellular proteases as activators. Several in vitro models have been developed to study the CoVs entry, pathogenesis, and possible therapeutic approaches. This article is aimed at summarizing the current knowledge about the use of relevant methodologies and cell lines permissive for CoV life cycle studies. The synthesis of this information can be useful for setting up specific experimental procedures. We also discuss different strategies for inhibiting the binding of the S protein to the cell receptors and the fusion process which may offer opportunities for therapeutic intervention.

Functional Characterization and Proteomic Analysis of Porcine Deltacoronavirus Accessory Protein NS7.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in neonatal piglets. Like other coronaviruses, PDCoV encodes at least three accessory or species-specific proteins; however, the biological roles of these proteins in PDCoV replication remain undetermined. As a first step toward understanding the biology of the PDCoV accessory proteins, we established a stable porcine cell line constitutively expressing the PDCoV NS7 protein in order to investigate the functional characteristics of NS7 for viral replication. Confocal microscopy and subcellular fractionation revealed that the NS7 protein was extensively distributed in the mitochondria. Proteomic analysis was then conducted to assess the expression dynamics of the host proteins in the PDCoV NS7-expressing cells. Highresolution two-dimensional gel electrophoresis initially identified 48 protein spots which were differentially expressed in the presence of NS7. Seven of these spots, including two upregulated and five down-regulated protein spots, showed statistically significant alterations, and were selected for subsequent protein identification. The affected cellular proteins identified in this study were classified into functional groups involved in various cellular processes such as cytoskeleton networks and cell communication, metabolism, and protein biosynthesis. A substantial down-regulation of α-actinin-4 was confirmed in NS7-expressing and PDCoV-infected cells. These proteomic data will provide insights into the understanding of specific cellular responses to the accessory protein during PDCoV infection.

Characterization of a bafinivirus exoribonuclease activity.

White bream virus (WBV), a poorly characterized plus-strand RNA virus infecting freshwater fish of the Cyprinidae family, is the prototype species of the genus Bafinivirus in the subfamily Torovirinae (family Coronaviridae, order Nidovirales). In common with other nidoviruses featuring >20 kilobase genomes, bafiniviruses have been predicted to encode an exoribonuclease (ExoN) in their replicase gene. Here, we used information on the substrate specificity of bafinivirus 3C-like proteases to express WBV ExoN in an active form in Escherichia coli. The 374-residue protein displayed robust 3'-to-5' exoribonuclease activity in the presence of Mg2+ ions and, unlike its coronavirus homologues, did not require a protein cofactor for activity. Characterization of mutant forms of ExoN provided support for predictions on putative active-site and conserved zinc-binding residues. WBV ExoN was revealed to be most active on double-stranded RNA substrates containing one or two non-paired 3'-terminal nucleotides, supporting its presumed role in increasing the fidelity of the bafinivirus RNA-dependent RNA polymerase.

Antiviral activity of K22 against members of the order Nidovirales.

Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 µM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein.

The multi-domain non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome, with an average molecular mass of about 200 kD. Nsp3 is an essential component of the replication/transcription complex. It comprises various domains, the organization of which differs between CoV genera, due to duplication or absence of some domains. However, eight domains of Nsp3 exist in all known CoVs: the ubiquitin-like domain 1 (Ubl1), the Glu-rich acidic domain (also called "hypervariable region"), a macrodomain (also named "X domain"), the ubiquitin-like domain 2 (Ubl2), the papain-like protease 2 (PL2pro), the Nsp3 ectodomain (3Ecto, also called "zinc-finger domain"), as well as the domains Y1 and CoV-Y of unknown functions. In addition, the two transmembrane regions, TM1 and TM2, exist in all CoVs. The three-dimensional structures of domains in the N-terminal two thirds of Nsp3 have been investigated by X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy since the outbreaks of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2003 as well as Middle-East Respiratory Syndrome coronavirus (MERS-CoV) in 2012. In this review, the structures and functions of these domains of Nsp3 are discussed in depth.

Detecting respiratory viral RNA using expanded genetic alphabets and self-avoiding DNA.

Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by "transliteration" technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-µl sample.

Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. Like other coronaviruses, PDCoV commonly contains 4 major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Among these, the N protein is known to be the most abundant and multifunctional viral component. Therefore, as the first step toward understanding the biology of PDCoV, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the PDCoV N protein. Similar to N proteins of other coronaviruses, the PDCoV N protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. We then assessed alterations in production levels of proteins in the N-expressing PK (PK-PDCoV-N) cells at different time points by means of proteomic analysis. According to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in PK-PDCoV-N cells in comparison with control PK cells. Of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. Notably, two members of the heat shock protein 70 family were found to be up-regulated in PK-PDCoV-N cells. These proteomic data will provide insights into the specific cellular response to the N protein during PDCoV infection.

Autophagy-independent LC3 function in vesicular traffic.

As protein folding is an imperfect process, the endoplasmic reticulum (ER) contains folding as well as ER-associated degradation (ERAD) machineries. In order to prevent premature interruption of folding, ERAD regulators and effectors such as EDEM1 and OS-9 are selectively cleared from the ER in so-called EDEMosomes to downregulate the degradative activity. The mechanism by which EDEM1 and OS-9 are subjected to rapid turnover, also known as ERAD tuning, shows similarities with, but is clearly distinct from, macroautophagy. Positive strand RNA coronaviruses (CoVs) such as the severe acute respiratory syndrome (SARS)-CoV and mouse hepatitis virus (MHV), induce in infected cells the formation of autophagosome-like, double-membrane vesicles (DMVs) to which their replication and transcription complexes are anchored. While it seems clear that CoVs hijack ER-derived host cell membranes for replication, the mechanism by which these DMVs are assembled has remained completely mysterious.

Identification of the murine coronavirus MP1 cleavage site recognized by papain-like proteinase 2.

The replicase polyprotein of murine coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a trans-cleavage assay and showed that PLP2 cleaves the replicase polyprotein between p210 and membrane protein 1 (MP1) (A. Kanjanahaluethai and S. C. Baker, J. Virol. 74:7911-7921, 2000). Here, we report the results of our studies identifying and characterizing this cleavage site. To determine the approximate position of the cleavage site, we expressed constructs that extended various distances upstream from the previously defined C-terminal end of MP1. We found that the construct extending from the putative PLP2 cleavage site at glycine 2840-alanine 2841 was most similar in size to the processed MP1 replicase product generated in a trans-cleavage assay. To determine which amino acids are critical for PLP2 recognition and processing, we generated 14 constructs with amino acid substitutions upstream and downstream of the putative cleavage site and assessed the effects of the mutations in the PLP2 trans-cleavage assay. We found that substitutions at phenylalanine 2835, glycine 2839, or glycine 2840 resulted in a reduction in cleavage of MP1. Finally, to unequivocally identify this cleavage site, we isolated radiolabeled MP1 protein and determined the position of [(35)S]methionine residues released by Edman degradation reaction. We found that the amino-terminal residue of MP1 corresponds to alanine 2841. Therefore, murine coronavirus PLP2 cleaves the replicase polyprotein between glycine 2840 and alanine 2841, and the critical determinants for PLP2 recognition and processing occupy the P6, P2, and P1 positions of the cleavage site. This study is the first report of the identification and characterization of a cleavage site recognized by murine coronavirus PLP2 activity.

Characterisation and mutational analysis of an ORF 1a-encoding proteinase domain responsible for proteolytic processing of the infectious bronchitis virus 1a/1b polyprotein.

Coronavirus gene expression involves proteolytic processing of the mRNA 1-encoded polyproteins by viral and cellular proteinases. Recently, we have demonstrated that an ORF 1b-encoded 100-kDa protein is proteolytically cleaved from the 1a/1b fusion polyprotein by a viral-specific proteinase of the picornavirus 3C proteinase group (3C-like proteinase). In this report, the 3C-like proteinase has been further analysed by internal deletion of a 2.3-kb fragment between the 3C-like proteinase-encoding region and ORF 1b and by substitution mutations of its catalytic centre as well as the two predicted cleavage sites flanking the 100-kDa protein. The results show that internal deletion of ORF 1a sequences from nucleotide 9911 to 12227 does not influence the catalytic activity of the proteinase in processing of the 1a/1b polyprotein to the 100-kDa protein species. Site-directed mutagenesis studies have confirmed that the predicted nucleophilic cysteine residue (Cys2922) and a histidine residue encoded by ORF 1a from nucleotide 8985 to 8987 (His2820) are essential for the catalytic activity of the proteinase, and that the QS(G) dipeptide bonds are its target cleavage sites. Substitution mutations of the third component of the putative catalytic triad, the glutamic acid 2843 (Glu2843) residue, however, do not affect the processing to the 100-kDa protein. In addition, cotransfection experiment shows that the 3C-like proteinase is capable of trans-cleavage of the 1a/1b polyprotein. These studies have confirmed the involvement of the 3C-like proteinase domain in processing of the 1a/1b polyprotein, the predicted catalytic centre of the proteinase, and its cleavage sites.

Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding.

The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.

Structural and functional analysis of the surface protein of human coronavirus OC43.

The two surface glycoproteins S and HE of human coronavirus OC43 (HCV-OC43) were isolated from the viral membrane and purified. Only the S protein was able to agglutinate chicken erythrocytes, indicating that this viral protein is the major hemagglutinin of HCV-OC43. The receptor determinant recognized by this virus on the surface of erythrocytes is N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) which is also used by bovine coronavirus for attachment to cells. By analyzing erythrocytes containing different amounts of Neu5,9Ac2 in either of two linkage types, it was found that there are subtle differences in the affinity of both viruses for 9-O-acetylated sialic acid. Bovine coronavirus was more efficient in recognizing low amounts of Neu5,9Ac2 alpha 2,3 linked to galactose, whereas HCV-OC43 was superior with respect to the alpha 2,6 linkage. The gene coding for the S protein of HCV-OC43 was cloned and sequenced. A large open reading frame predicts a polypeptide of 150 kDa in the unglycosylated form. A protein of about 190 kDa is expected if the 20 potential glycosylation sites are used for attachment of N-linked oligosaccharide side chains. These predictions were confirmed by in vitro transcription and translation of the gene in the presence or absence of canine pancreatic microsomal membranes. A high degree of sequence homology was found between the S proteins of HCV-OC43 and bovine coronavirus. Structural and functional analyses of more strains should help to identify the location of the sialic acid-binding site.

Fusion formation by the uncleaved spike protein of murine coronavirus JHMV variant cl-2.

The fusogenic properties of the uncleaved spike (S) protein of murine coronavirus JHMV variant cl-2 were studied by expressing the S protein with a deleted putative cleavage site. The amino acid sequence of the putative cleavage site, Arg-Arg-Ala-Arg-Arg, was replaced by Arg-Thr-Ala-Leu-Glu by in vitro mutagenesis of the cl-2 S protein cDNA. Recombinant vaccinia viruses containing the cl-2 S cDNA [RVV t(+)] or the mutated cDNA [RVV t(-)] were constructed and monitored for fusion formation and cleavage of the expressed S proteins. When cultured DBT cells were infected with RVV t(+) at a multiplicity of infection of 0.5, fusion formation was first observed at 10 to 12 h postinoculation and spread throughout the whole culture by 20 to 24 h postinoculation. In cells infected with RVV t(-) under the same conditions, fusion formation appeared by 12 to 14 h. This result represented a 2- to 4-h delay in the onset of fusion, compared with its appearance in cells expressing the wild-type S protein. By 25 to 30 h, most of the cells infected by RVV t(-) had fused. By immunoprecipitation and Western blotting (immunoblotting), the 170-kDa S protein was detected in DBT cells expressing the wild-type S protein and the mutated S protein. However, interestingly, the cleavage products of the S protein, S1 and S2, were not detected in RVV t(-)-infected cells, producing the mutated S protein, even though fusion was clearly visible. Both products were, of course, detected in RVV t(+)-infected DBT cells, producing the wild-type S protein. The same results concerning the fusion formation and cleavage properties of the S proteins were reproduced by the transiently expressed S proteins. These results suggest that the cleavage event in the S protein of murine coronavirus JHMV is not a prerequisite for fusion formation but that it does facilitate fusion formation.

Cell biology of viruses that assemble along the biosynthetic pathway.

In this review we discuss five groups of viruses that bud into, or assemble from, different compartments along the biosynthetic pathway. These are herpes-, rota-, corona-, bunya- and pox-viruses. Our main emphasis will be on the virally-encoded membrane glycoproteins that are responsible for determining the site of virus assembly. In a number of cases these proteins have been well characterized and appear to serve as resident markers of the budding compartments. The assembly and dissemination of these viruses raises many questions of cell biological interest.

Membrane assembly of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation.

The M protein of mouse hepatitis virus strain A59 is a triple-spanning membrane protein which assembles with an uncleaved internal signal sequence, adopting an NexoCcyt orientation. To study the insertion mechanism of this protein, domains potentially involved in topogenesis were deleted and the effects analyzed in topogenesis were deleted and the effects analyzed in several ways. Mutant proteins were synthesized in a cell-free translation system in the presence of microsomal membranes, and their integration and topology were determined by alkaline extraction and by protease-protection experiments. By expression in COS-1 and Madin-Darby canine kidney-II cells, the topology of the mutant proteins was also analyzed in vivo. Glycosylation was used as a biochemical marker to assess the disposition of the NH2 terminus. An indirect immunofluorescence assay on semi-intact Madin-Darby canine kidney-II cells using domain-specific antibodies served to identify the cytoplasmically exposed domains. The results show that each membrane-spanning domain acts independently as an insertion and anchor signal and adopts an intrinsic preferred orientation in the lipid bilayer which corresponds to the disposition of the transmembrane domain in the wild-type assembled protein. These observations provide further insight into the mechanism of membrane integration of multispanning proteins. A model for the insertion of the coronavirus M protein is proposed.

Synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the E3 region of adenovirus.

The haemagglutinin-esterase gene (HE) of bovine coronavirus (BCV) encodes a major viral membrane glycoprotein that elicits BCV-neutralizing antibodies. The BCV HE gene was cloned into a human adenovirus serotype 5 (Ad5) transfer vector in place of early transcription region 3, and a helper-independent recombinant virus was constructed by rescue of the transcription unit by homologous in vivo recombination between the vector and Ad5 genomic DNA. The BCV HE polypeptide expressed by this recombinant Ad was characterized in vivo and in vitro. A 65K polypeptide was identified using an anti-BCV antibody in both human (293) and bovine (MDBK) cells infected with the recombinant Ad. In the absence of a reducing agent, migration of the 65K polypeptide was shifted to 130K, indicating that the recombinant HE polypeptide existed in a dimeric form. The HE polypeptide was glycosylated, as demonstrated by labelling with [3H]glucosamine, and was immunoreactive with three distinct groups of conformation-specific anti-HE monoclonal antibodies (MAbs). Cells infected with recombinant Ad expressing BCV HE exhibited both haemadsorption activity and acetylesterase activity. In addition, the anti-HE group A MAbs HC10-5 and KD9-40 inhibited both the haemadsorption activity and esterase activity of the recombinant HE polypeptide, suggesting that the antigenic domain responsible for BCV neutralization may overlap (or is closely associated with) the domain(s) responsible for haemagglutination and/or acetylesterase activities. When mice were inoculated intraperitoneally with live recombinant Ad, a significant level of BCV-neutralizing HE-specific antibody was induced. These results indicate that the recombinant Ad replicates and directs the synthesis of the BCV HE polypeptide in vivo.

Sequence analysis of the membrane protein gene of human coronavirus OC43 and evidence for O-glycosylation.

The gene encoding the membrane (M) protein of the OC43 strain of human coronavirus (HCV-OC43) was amplified by a reverse transcription-polymerase chain reaction of viral RNA with HCV-OC43- and bovine coronavirus (BCV)-specific primers. The nucleotide sequence of the cloned 1.5 kb fragment revealed an open reading frame (ORF) of 690 nucleotides which was identified as the M protein gene from its homology to BCV. This ORF encodes a protein of 230 amino acids with an M(r) of 26416. The gene is preceded by the motif UCCAAAC, analogous to the consensus coronavirus transcription initiation sequence. The M protein of HCV-OC43 shows features typical of all coronavirus M proteins studied: a hydrophilic, presumably external N terminus including about 10% of the protein, and a potential N-glycosylation site followed by three major hydrophobic transmembrane domains. The amino acid sequence of the M protein of HCV-OC43 has 94% identity with that of the Mebus strain of BCV, and also contains six potential O-glycosylation sites in the exposed N-terminal domain. Indeed, the glycosylation of the M protein was not inhibited in the presence of tunicamycin, which is indicative of O-glycosylation, as previously reported for BCV and murine hepatitis virus. Virions released from tunicamycin-treated cells contained the M glycoprotein but were devoid of both peplomer (S) and haemagglutinin-esterase (HE) proteins. Thus, inhibition of the N-glycosylation of the S and HE structural proteins prevented their incorporation into progeny virions, an indication that they are dispensable for virion morphogenesis, unlike the M protein.