Coronavirus hemagglutinin-esterase and spike proteins coevolve for functional balance and optimal virion avidity.

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

Crystal structure of bovine coronavirus spike protein lectin domain.

The spike protein N-terminal domains (NTDs) of bovine coronavirus (BCoV) and mouse hepatitis coronavirus (MHV) recognize sugar and protein receptors, respectively, despite their significant sequence homology. We recently determined the crystal structure of MHV NTD complexed with its protein receptor murine carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which surprisingly revealed a human galectin (galactose-binding lectin) fold in MHV NTD. Here, we have determined at 1.55 Å resolution the crystal structure of BCoV NTD, which also has the human galectin fold. Using mutagenesis, we have located the sugar-binding site in BCoV NTD, which overlaps with the galactose-binding site in human galectins. Using a glycan array screen, we have identified 5-N-acetyl-9-O-acetylneuraminic acid as the preferred sugar substrate for BCoV NTD. Subtle structural differences between BCoV and MHV NTDs, primarily involving different conformations of receptor-binding loops, explain why BCoV NTD does not bind CEACAM1 and why MHV NTD does not bind sugar. These results suggest a successful viral evolution strategy in which coronaviruses stole a galectin from hosts, incorporated it into their spike protein, and evolved it into viral receptor-binding domains with altered sugar specificity in contemporary BCoV or novel protein specificity in contemporary MHV.

Bovine coronavirus I protein synthesis follows ribosomal scanning on the bicistronic N mRNA.

The mRNA encoding the 49-kDa nucleocapsid protein (N) of the bovine coronavirus is bicistronic. A 23-kDa protein, termed the I protein for the 'internal' open reading frame (ORF), is also synthetized but in the +1 reading frame beginning 61 nt downstream of the N start codon. Sequences flanking the N and I start codons suggest that the I ORF might be accessed by scanning ribosomes passing over the N start codon. Here we test this idea and demonstrate with translation studies both in vitro and in vivo that the I protein is synthesized according to the leaky scanning model for initiation of translation on the subgenomic N mRNA molecule.

Recognition of cellular receptors by bovine coronavirus.

Bovine coronavirus (BCV) initiates infection by attachment to cell surface receptors the crucial component of which is N-acetyl-9-O-acetylneuraminic acid. Inactivation of receptors by neuraminidase treatment and restoration of receptors by enzymatic resialylation of asialo-cells is described as a method to determine (i) the type of sialic acid that is recognized; (ii) the linkage specificity of the viral binding activity; (iii) the minimal amount of sialic acid required for virus attachment. Evidence is presented that both glycoproteins and glycolipids can serve as receptors for BCV provided they contain 9-O-acetylated sialic acid. A model is introduced proposing that after initial binding to sialic acid-containing receptors, the S-protein of BCV interacts with a specific protein receptor. This interaction may result in a conformational change that exposes a fusogenic domain and thus induces the fusion between the viral and the cellular membrane.

Coronavirus nucleocapsid protein. RNA interactions.

The coronavirus nucleocapsid protein (N) is involved in encapsidation and packaging of viral RNA. In this study we investigated the ability of the bovine coronavirus (BCV) N protein to interact with RNA. Histidine-tagged BCV N (his-N) protein was expressed in bacteria. A filter binding assay was established to quantitatively measure the binding efficiency of purified his-N to different RNAs. The results indicate that bacterially expressed N bound both BCV and mouse hepatitis coronavirus (MHV) RNAs. Binding to in vitro generated BCV and MHV RNA transcripts was significantly higher than binding to a non-coronavirus RNA. Similar binding efficiencies were measured for a BCV defective genome, pDrep, and a transcript that contained the MHV packaging signal. Interestingly, the entire MHV DI, pMIDI-C, was bound at a higher efficiency than the packaging signal alone. This is one of the first reports to show that N interacts with the MHV packaging signal.

Bovine coronavirus nucleocapsid protein processing and assembly.

The coronavirus nucleocapsid protein (N) encapsidates the genomic RNA to form a helical nucleocapsid. The requirements for coronavirus nucleocapsid assembly are being studied. Two forms (approximately 50 kDa and 55 kDa) of the bovine coronavirus (BCV) N protein were detected in infected cells. However, only one form, a 50 kDa species, was detected in extracellular virions. After treatment with calf intestinal alkaline phosphatase (CIAP), the 55 kDa intracellular form increased in mobility to comigrate with the 50 kDa form; whereas, the 50 kDa intracellular species and N from extracellular virions was not sensitive to CIAP treatment. The data indicate that specificity exists with regard to assembly of N into the mature virion. The data suggests that processing of N may take place during assembly of either nucleocapsids or virions and that the processing may be a dephosphorylation event.

Evidence for a pseudoknot in the 3' untranslated region of the bovine coronavirus genome.

A potential pseudoknot was found in the 3' untranslated region of the bovine coronavirus genome beginning 63 nt downstream from the stop codon of the N gene. Mutation analysis of the pseudoknot in a cloned defective interfering RNA indicated that this structural element is necessary for defective interfering RNA replication.

Coronavirus envelope glycoprotein assembly complexes.

Protein:protein interactions, and their subcellular localization, play important roles in coronavirus assembly. In this study, we have identified similar envelope glycoprotein complexes that are present in mouse hepatitis coronavirus A59 (MHV-A59) and bovine coronavirus (BCV) infected cells. Complexes consisting of the spike (S) and membrane (M) proteins were identified in cells infected with MHV-A59 or BCV. Kinetic analyses demonstrated that S and M quickly associated after translation, and suggested that both initially interacted in a pre-Golgi site. In addition, the hemagglutinin esterase (HE) was identified as part of a complex with M and S in BCV infected cells. Taken together, our data indicate that similar glycoprotein complexes are present in cells infected with two different coronaviruses, and thus likely represent important prerequisite complexes involved in virus assembly.

Recognition of N-acetyl-9-O-acetylneuraminic acid by bovine coronavirus and hemagglutinating encephalomyelitis virus.

The S protein of hemagglutinating encephalomyelitis virus is shown to be a hemagglutinin requiring N-acetyl-9-O-acetylneuraminic acid as a receptor determinant on the surface of erythrocytes. The ability of bovine coronavirus to recognize 9-O-acetylated sialic acid was used to establish a binding assay for the detection of glycoproteins containing this type of sugar. The assay is very fast, because it uses the acetylesterase of the viral HE protein to localize bound virus.

Negatively charged residues in the endodomain are critical for specific assembly of spike protein into murine coronavirus.

Coronavirus spike (S) protein assembles into virions via its carboxy-terminus, which is composed of a transmembrane domain and an endodomain. Here, the carboxy-terminal charge-rich motif in the endodomain was verified to be critical for the specificity of S assembly into mouse hepatitis virus (MHV). Recombinant MHVs exhibited a range of abilities to accommodate the homologous S endodomains from the betacoronaviruses bovine coronavirus and human SARS-associated coronavirus, the alphacoronavirus porcine transmissible gastroenteritis virus (TGEV), and the gammacoronavirus avian infectious bronchitis virus respectively. Interestingly, in TGEV endodomain chimeras the reverting mutations resulted in stronger S incorporation into virions, and a net gain of negatively charged residues in the charge-rich motif accounted for the improvement. Additionally, MHV S assembly could also be rescued by the acidic carboxy-terminal domain of the nucleocapsid protein. These results indicate an important role for negatively charged endodomain residues in the incorporation of MHV S protein into assembled virions.

The detection and genetic characterization based on the S1 gene region of BCoVs from respiratory and enteric infections in Turkey.

We investigated bovine coronavirus (BCoV) as an etiological agent in cattle with clinical respiratory and digestive signs using 147 feces and 199 nasal swab samples. A total of 18 test samples (16 feces and 2 nasal swap samples) were detected positive by ELISA and/or RT-PCR targeting the BCoV N gene. The partial S1 gene regions of BCoVs (An-4 and An-11) detected in feces samples from two herd-mate dairy calves were compared. Virological and serological results indicated that BCoVs are widespread in Turkey and are likely etiological agents in diarrhea cases in calves.

A translation-attenuating intraleader open reading frame is selected on coronavirus mRNAs during persistent infection.

Short open reading frames within the 5' leader of some eukaryotic mRNAs are known to regulate the rate of translation initiation on the downstream open reading frame. By employing the polymerase chain reaction, we learned that the 5'-terminal 5 nt on the common leader sequence of bovine coronavirus subgenomic mRNAs were heterogeneous and hypervariable throughout early infection in cell culture and that as a persistent infection became established, termini giving rise to a common 33-nt intraleader open reading frame were selected. Since the common leader is derived from the genomic 5' end during transcription, a common focus of origin for the heterogeneity is expected. The intraleader open reading frame was shown by in vitro translation studies to attenuate translation of downstream open reading frames in a cloned bovine coronavirus mRNA molecule. Selection of an intraleader open reading frame resulting in a general attenuation of mRNA translation and a consequent attenuation of virus replication may, therefore, be a mechanism by which coronaviruses and possibly other RNA viruses with a similar transcriptional strategy maintain a persistent infection.

Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins.

Targeted RNA recombination was used to construct mouse hepatitis virus (MHV) mutants containing chimeric nucleocapsid (N) protein genes in which segments of the bovine coronavirus N gene were substituted in place of their corresponding MHV sequences. This defined portions of the two N proteins that, despite evolutionary divergence, have remained functionally equivalent. These regions included most of the centrally located RNA-binding domain and two putative spacers that link the three domains of the N protein. By contrast, the amino terminus of N, the acidic carboxy-terminal domain, and a serine- and arginine-rich segment of the central domain could not be transferred from bovine coronavirus to MHV, presumably because these parts of the molecule participate in protein-protein interactions that are specific for each virus (or, possibly, each host). Our results demonstrate that targeted recombination can be used to make extensive substitutions in the coronavirus genome and can generate recombinants that could not otherwise be made between two viruses separated by a species barrier. The implications of these findings for N protein structure and function as well as for coronavirus RNA recombination are discussed.

Stem-loop III in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication.

Higher-order structures in the 5' untranslated region (UTR) of plus-strand RNA viruses are known in many cases to function as cis-acting elements in RNA translation, replication, or transcription. Here we describe evidence supporting the structure and a cis-acting function in defective interfering (DI) RNA replication of stem-loop III, the third of four predicted higher-order structures mapping within the 210-nucleotide (nt) 5' UTR of the 32-kb bovine coronavirus (BCoV) genome. Stem-loop III maps at nt 97 through 116, has a calculated free energy of -9.1 kcal/mol in the positive strand and -3.0 kcal/mol in the negative strand, and has associated with it beginning at nt 100 an open reading frame (ORF) potentially encoding an 8-amino-acid peptide. Stem-loop III is presumed to function in the positive strand, but its strand of action has not been established. Stem-loop III (i) shows phylogenetic conservation among group 2 coronaviruses and appears to have a homolog in coronavirus groups 1 and 3, (ii) has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5' UTR ORF, (iii) is supported by enzyme structure-probing evidence in BCoV RNA, (iv) must maintain stem integrity for DI RNA replication in BCoV DI RNA, and (v) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation in BCoV DI RNA. These results indicate that stem-loop III in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that its associated intra-5' UTR ORF may function to enhance replication. It is postulated that these two elements function similarly in the virus genome.

A cis-acting function for the coronavirus leader in defective interfering RNA replication.

To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terminal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of terminal bases that destroyed a predicted intraleader stem-loop also destroyed replicating ability. Surprisingly, when the same stem-loop was disrupted by base substitutions, replication appeared only minimally impaired and the leader was found to have rapidly reverted to wild type during DI RNA replication, a phenomenon reminiscent of high-frequency leader switching in the mouse hepatitis coronavirus. These results suggest that once a minimal structural requirement for leader is fulfilled for initiation of DI RNA replication, the wild-type leader is strongly preferred for subsequent replication. They also demonstrate that, in contrast to reported natural mouse hepatitis coronavirus DI RNAs, the DI RNA of the bovine coronavirus does not require sequence elements originating from discontinuous downstream regions within the polymerase gene for replication or for packaging.

Protein interactions during coronavirus assembly.

Coronaviruses assemble and obtain their envelope at membranes of the intermediate compartment between the endoplasmic reticulum and Golgi complex. Like other enveloped viruses, coronavirus assembly is presumably dependent on protein localization and protein-protein as well as protein-RNA interactions. We have used the bovine coronavirus (BCV) as a model to study interactions between the viral proteins in virus-infected cells that are important for coronavirus assembly. BCV is a prototype for the coronaviruses that express an additional major structural protein, the hemagglutinin esterase (HE), in addition to the spike (S) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein. Complexes consisting of the M, S, and HE proteins were detected in virus-infected cells by coimmunoprecipitations. Kinetic analyses demonstrated that S protein and HE each quickly formed a complex with M protein after synthesis, whereas heterocomplexes consisting of all three proteins formed more slowly. The kinetics of HE biosynthesis revealed that the half-life of oligomerization was approximately 30 min, which correlated with the appearance of complexes consisting of M, HE, and S proteins, suggesting that oligomerization and/or conformational changes may be important for the S-M-HE protein complexes to form. Only HE dimers were found associated with the heterocomplexes consisting of all three proteins. S-M-HE protein complexes were detected prior to processing of the oligosaccharide chains on HE, indicating that these protein complexes formed in a premedial Golgi compartment before trimming of sugar chains. Transient coexpressions and double-labeling immunofluorescence demonstrated that HE and S proteins colocalized with M protein. This was further supported by coimmunoprecipitation of specific HE-M and S-M protein complexes from transfected cells, indicating that these proteins can form complexes in the absence of other viral proteins.

Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus.

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

The spike but not the hemagglutinin/esterase protein of bovine coronavirus is necessary and sufficient for viral infection.

The spike (S) and hemagglutinin/esterase (HE) of bovine coronavirus (BCV) are the two envelope proteins that recognize the same receptor-determinant of 9-O-acetylneuraminic acid on host cells. However, the precise and relative roles of the two proteins in BCV infectivity remain elusive. To unequivocally determine their roles in viral cytopathogenicity, we developed a system in which phenotypically chimeric viruses were generated by infecting a closely related mouse hepatitis virus (MHV) in cells that stably express an individual BCV protein (S or HE). The chimeric viruses were then used to infect human rectal tumor (HRT)-18 cells that are permissive to BCV but are nonsusceptible to MHV. Using this approach, we found that the chimeric virus containing the BCV S protein on the virion surface entered and replicated in HRT-18 cells; this was specifically blocked by prior treatment of the virus with a neutralizing antibody specific to the BCV S protein, indicating that the BCV S protein is responsible for initiating chimeric virus infection. In contrast, chimeric viruses that contain biologically active and functional BCV HE protein on the surface failed to enter HRT-18 cells, indicating that the BCV HE protein alone is not sufficient for BCV infection. Taken together, these results demonstrate that the S protein but not the HE protein of BCV is necessary and sufficient for infection of the chimeric viruses in HRT-18 cells, suggesting that BCV likely uses the S protein as a primary vehicle to infect permissive cells.