Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus

PV was obtained from P. A. Nuttall (NERC Oxford). MHV-S was kindly supplied by M. J. Buchmeier (Scripps Research Institute, La Jolla, Calif.). These viruses and MHV-A59 were grown in mouse L or DBT cells. Influenza C/JJ/50 virus was isolated from embryonated eggs as described earlier (33).

For isolation of genomic RNA, PV was concentrated from tissue culture supernatants by precipitation with polyethylene glycol and purified on a sucrose gradient (35). Genomic and intracellular RNA from infected cells was isolated as described previously (30).

For labeling viral proteins, L cells infected with PV (multiplicity of infection of 2) were incubated at 10 h postinfection (p.i.) with labeling medium containing 200 μCi of ³⁵S-Translabel (ARC, St. Louis, Mo.) per ml. After incubation for 3 h at 37°C, virus particles were purified from the supernatant by centrifugation on a 20 to 60% sucrose step gradient. Virus collected from the interphase was pelleted by ultracentrifugation and analyzed by sodium dodecyl sulfate (SDS)–10% polyacrylamide gel electrophoresis (PAGE). For radioimmunoprecipitation analysis, labeled cell lysates were collected 10 h p.i., incubated with rabbit antiserum specific for MHV-JHM (kindly provided by J. Thalhammer, University of Salzburg), and analyzed as described previously (33).

Purified RNA was reverse transcribed (8) with Superscript II reverse transcriptase (Gibco) by using random hexanucleotides (Pharmacia). EcoRI linkers were ligated to double-stranded cDNA. Random-primed cDNA was ligated into the EcoRI site of pBluescript (Promega) and transformed into Escherichia coli DH5α by standard procedures (24). Nucleotide sequence analysis was performed with an automated sequencer (LICOR) on both strands of the cloned cDNA. The nucleotide sequence data presented here were submitted to the EBI/EMBL database and are available under accession number AJ005960 .

Acetylesterase activity with p -nitrophenylacetate ( p NPA) as the substrate was determined as described previously (33). One unit was defined as the amount of enzymatic activity resulting in the cleavage of 1 μmol of p NPA per min.

Release of acetate from glycoconjugates was determined with a commercial test kit as described earlier (35). Acetate contents of sialoconjugates were determined by saponification in 0.2 M NaOH at room temperature and subsequent neutralization with 0.2 M HCl (44). BSM types I and I-S, porcine mucin type III, calf fetuin, and bovine brain extract type VI were obtained from Sigma-Aldrich. Mouse α₁ macroglobulin was purified from mouse serum similar to published procedures (10, 15) with the following modifications: purification involved precipitation with 12% polyethylene glycol 6000 and sequential chromatography on Blue Sepharose CL6B and HiTrap Q Sepharose connected to an ÄKTA purifier (Pharmacia). Hemagglutinin (HA) inhibition activity of fractions obtained was determined with influenza C/JJ/50 virus.

In situ staining of virus plaques was performed with α-naphthyl acetate (αNA) with a cytochemical esterase staining kit (Sigma). Approximately 48 h p.i., cells were fixed for 4 h by adding CAF solution (4.6 mM citric acid, 2.3 mM Na citrate, 3 mM NaCl, 66% acetone, 3% formaldehyde [pH 3.6]) on top of the agarose overlay. After removal of the agarose, cells were washed with H₂O. Esterase-expressing viral plaques were detected by incubation with αNA-Fast Blue BB solution for 15 to 30 min at 37°C according to the manufacturer’s instructions. Reactions were stopped by washing the cells with H₂O.

HA and HI assays were performed as described previously (35) with 0.5% human erythrocytes obtained from the local blood bank. HA titers were expressed as the reciprocal of highest virus dilution resulting in full agglutination of erythrocytes.

Virus binding assays were performed on coated 96-well microtiter plates as described elsewhere (44). Glycoproteins were dissolved in phosphate-buffered saline (PBS) and allowed to bind at 4°C overnight (50 μl/well). Bovine brain extract type VI was dissolved in methanol, added to microtiter wells (50 μl/well), and evaporated. Wells were then washed with PBS, and the remaining binding sites were blocked with 3% bovine serum albumin (BSA) in PBS for 2 h at room temperature. After removal of BSA, wells were washed with PBS and virus suspensions were added (50 μl/well) and incubated for 2 h at 4°C. After removal of virus, wells were washed three times with PBS. Bound virus was detected by incubation with the synthetic substrate 4-methylumbelliferyl acetate (4MUAc). A 5 mM stock solution (in acetone) was diluted 50-fold with PBS, and 100 μl was added to the microtiter wells and incubated at 37°C. Cleavage of substrate was monitored at an excitation wavelength of 365 nm.

For further characterization of this virus, we first isolated RNA from infected cells. To determine the number of subgenomic RNAs transcribed from the genome of this virus, total RNA was subjected to electrophoresis on a denaturing agarose gel and hybridized with oligonucleotide O48 (5′-GTGATTCTTCCAATTGGCCATG-3′) complementary to a conserved region at the 3′ end of MHV and related viruses. Compared to MHV-A59 an additional RNA was found in cells infected with PV, migrating slightly faster than mRNA 2 (Fig. 1). A similar mRNA 2-1 encoding the HE protein is present in several MHV strains (28). The presence of this mRNA does not necessarily indicate expression of the HE protein. Due to point mutations or deletions in the coding region of the HE gene, several MHV strains do not express a functional HE (40). We therefore investigated whether PV does express this protein. Initial tests with the synthetic esterase substrate p NPA indicated relatively low levels of acetylesterase activity in virus preparations (data not shown). This might have been due to the low expression rates of the HE gene or to the presence of a mixture of viruses with or without a functional gene. We therefore plaque purified PV and screened individual isolates for acetylesterase activity. Among 24 preparations, isolates PV5 and PV14 were identified as expressing acetylesterase activity. To identify esterase-expressing virus plaques, we used an in situ staining procedure with αNA. This substrate has been used earlier to detect esterase activity of influenza C virus in infected MDCK cells (37) or immobilized on nitrocellulose filters and thin-layer plates (44). We have extended this method to detect coronavirus esterases in infected cells. Plaques of isolate PV14 were stained due to the esterase activity yielding insoluble α-naphthol–Fast Blue BB precipitate on infected cells (Fig.2). As a negative control we used MHV-A59, which is devoid of HE protein (16). Unstained plaques were observed after incubation with αNA. For a positive control MHV-S, a strain expressing high levels of HE protein (39), was used in the assay. Other plaque-purified PV isolates exhibited no esterase activities. Thus, the initial low levels of esterase activity were caused by the presence of a mixed population. In the original PV preparation less than 10% of viruses expressed an active acetylesterase. PV14 exhibited acetylesterase activity comparable to that of MHV-S. By employing p NPA as a substrate, specific esterase activities of gradient-purified PV14 and MHV-S were found to be 5.9 and 6.7 mU/10⁶ PFU, respectively.

To determine expression rates and the apparent molecular weight of PV14 proteins, we prepared³⁵S-labeled virus and subjected it to SDS-PAGE (Fig.3A). The spike protein was found partially as uncleaved protein (S/gp180) and mainly as cleaved protein (S/gp90). In addition, the nucleoprotein (N) and at least three forms of the matrix protein (M) were clearly detectable. In contrast, the HE protein was found only in minor amounts after radioimmunoprecipitation with MHV-specific antiserum (Fig. 3B). Thus, expression rates of PV HE are comparable to those of MHV-JHM (29) and are lower than those described for MHV strains S and JHM(2) (39).

For further characterization, we cloned the HE gene of PV, which was isolated from a set of clones reverse transcribed with random primers. Sequence analysis revealed that this gene is related to that of MHV. The PV HE gene encodes a protein with 439 amino acid residues, including a 24-amino-acid signal sequence. The putative active site serine residue of viral acetylesterases (36) within the conserved FGDS sequence is present at position 45 (Fig. 4). In the PV sequence, 85 to 87% of the amino acid sequences are identical, whereas 59 to 65 residues are different from the HE proteins of MHV strains. The mature PV HE protein contains 11 asparagine residues potentially serving as glycosylation sites. The corresponding proteins of MHV possess 9 (MHV-JHM) or 10 glycosylation sites (MHV-S and MHV-DVIM). All cysteine residues present in the mature PV protein are at the same positions as those of the MHV HE proteins (Fig.5).

We then wanted to confirm that PV expresses sialate-9-O-acetylesterase activity. We first tested enzymatic activity with the synthetic substrate p NPA, and comparable rates of hydrolysis were observed for PV14 and influenza C/JJ/50 virus. To our surprise, we were unable to detect the release of acetate from BSM (Fig.6). On the other hand, control assays indicated that this substrate was readily cleaved by the influenza C/JJ/50 virus esterase. In this assay, we used BSM with a sialic acid content of 12% and an acetate content of 1.6%. Similar results were obtained with another BSM preparation with 5% sialic acid and a 1.2% acetate content. Since other coronaviruses, including BCV (35, 20, 42), and hemagglutinating encephalomyelitis virus (27) are known to cleave O -acetylated sialic acids on mucin, these findings were unexpected. We then investigated a possible reason for observed differences in the enzymatic activities of influenza C/JJ/50 and PV. By using p NPA as a substrate, pH optima of PV and influenza C/JJ/50 esterase were determined and found to range between pH 7.4 and 7.8 (data not shown). Thus, the inability of PV esterase to release acetate from BSM due to a different pH optimum can be ruled out. This led us to speculate that the substrate specificity of PV esterase might be different from those of BCV and influenza C viruses.

We then tested whether PV can remove receptors for influenza C virus from erythrocytes. Human erythrocytes were incubated with influenza C/JJ/50 virus, PV, or PBS. Then cells were washed three times with PBS and used for hemagglutination. Mock-treated or untreated erythrocytes were agglutinated by influenza C/JJ/50 virus (HA titer = 128). As expected (35), cells treated with influenza C virus were not agglutinated. In contrast, cells incubated with PV were agglutinated by C/JJ/50 with the same titer as the mock-treated cells. These data strongly indicate that Neu5,9Ac₂, a major receptor determinant for influenza C viruses (23), is not a substrate for PV acetylesterase.

We then tested whether human erythrocytes or erythrocytes from mouse strains BALB/c and C57BL were agglutinated by PV at neutral pH and at pH 6.5. Under these conditions, MHV-DVIM was shown to agglutinate murine and rat erythrocytes (31). PV exhibited no agglutinin activity under the conditions tested. Due to this lack of HA activity, experiments to remove PV receptors from erythrocytes could not be performed. We therefore used a solid-phase assay on microtiter plates, which allows detection of virus binding to sialoconjugates (44). In this assay, bound virus is detected by its acetylesterase activity, which converts 4MUAc to 4-methyl umbelliferone, a fluorescent dye. First, we tested whether PV was able to cleave 4MUAc. Serial dilutions of PV or influenza C/JJ/50 virus were incubated in microtiter wells with 0.1 mM 4MUAc at 37°C, and cleavage of the substrate was monitored at 365 nm. Both viruses were able to cleave this substrate at comparable rates (Fig.7A). We then coated microtiter wells with glycoconjugates and tested the binding of viruses. Of several glycoproteins tested in this assay, influenza C/JJ/50 virus exhibited strong binding activity towards bovine mucin, as expected from the esterase assays described above. In addition, murine α₁macroglobulin exhibited binding activity towards C/JJ/50. No binding of influenza C virus was observed in wells coated with porcine mucin and, as expected (44), with calf fetuin. In contrast, PV did not bind to any of these glycoproteins under the conditions tested. It should be mentioned that compared to C/JJ/50 virus, a 3.4-fold-higher concentration of PV, as calculated from esterase activities, was applied to coated microtiter wells. When we used even higher concentrations of PV, some binding activity with porcine mucin type III was detectable (data not shown). Since porcine mucin is believed to be devoid of O -acetylated sialic acids (18), we are currently investigating whether this observation is due to nonspecific interactions. In any case, we were unable to detect boundO-acetyl groups in porcine mucin after saponification. In addition to 9- O -acetylated glycoproteins, other glycoconjugates, such as gangliosides, can serve as influenza C virus receptors. Bovine brain gangliosides have been used to restore susceptibility to infection by influenza C virus of sialidase-treated MDCK cells (11). For this reason, we also tested the binding of viruses to bovine brain extract containing a mixture of phospholipids and glycolipids, including gangliosides. Again, the binding of influenza C/JJ/50 virus was observed but no reaction of PV with bovine brain extract was detectable (Fig. 7B).