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. 1999 May;73(5):3737-43.

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

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

A Klausegger et al. J Virol. .
Free PMC article

Abstract

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.

Figures

FIG. 1
FIG. 1
Hybridization analysis of viral mRNA. Intracellular RNA was isolated from infected DBT cells at 8 h p.i. and subjected to electrophoresis on a denaturing agarose gel. The dried gel was hybridized to 32P-labelled oligonucleotide O48 and autoradiographed. (A) MHV-A59-infected cells. (B) PV-infected cells. The numbers of viral mRNAs are indicated at the left, and mRNA 2-1 is indicated by an arrowhead.
FIG. 2
FIG. 2
In situ detection of acetylesterase activity in coronavirus plaques. L cells infected with coronavirus (20 to 100 PFU/dish) were fixed 36 h p.i. and stained with αNA for 15 to 30 min. (A) Plaque-purified isolate PV14. (B) MHV-A59. (C) MHV-S. Examples of unstained MHV-A59 plaques are marked with arrows.
FIG. 3
FIG. 3
Analysis of PV14 proteins. (A) L cells infected with PV14 were labelled 10 h p.i. with 35S-Translabel for 3 h. Virus particles were purified from the supernatant by centrifugation on a 20 to 60% sucrose step gradient, pelleted, and analyzed by SDS–10% PAGE. (B) Radioimmunoprecipitation of PV14 proteins. Infected L cells were labelled 8 h p.i. with 35S-Translabel and [3H]leucine for 3 h. Then cells were lysed, and viral proteins were precipitated with rabbit anti-MHV-JHM and subjected to SDS-PAGE. Positions of the matrix protein (M), nucleoprotein (N), HE, and two forms of spike protein (S/gp90 and S/gp180) are indicated by arrows. Positions of the marker proteins (in kilodaltons) are also indicated.
FIG. 4
FIG. 4
Nucleotide sequence of the HE gene of PV. The stop codon of the upstream gene coding for the nonstructural protein 2a and the initiation codon of the downstream spike gene are indicated by <<< and >>>, respectively. Intergenic promoter sequences are double underlined, and the stop codon of the HE gene is indicated by asterisks. The deduced amino acid sequence is shown in the one-letter code. The predicted N-terminal signal sequence and the presumptive C-terminal transmembrane region are underlined. The conserved FGDS sequence with the putative active site serine residue is shown in italics. Potential N-glycosylation sites are boxed.
FIG. 5
FIG. 5
Sequence alignment of the HE protein of PV with the corresponding proteins of MHV-DVIM (accession number PID g2662175), MHV-JHM (PID g543553), MHV-S (PID g555242), and BCV-Mebus (PID g122851). Amino acid residues identical to the PV sequence are shown as dashes; gaps introduced to allow optimal alignment are shown as dots. The putative catalytic site is underlined, potential glycosylation sites are shown double underlined. Cysteine residues are marked with an asterisk.
FIG. 6
FIG. 6
Acetate release from BSM. A total of 2.5 mU of purified PV or influenza C/JJ/50 virus was incubated with BSM (12.5 mg/ml) at 37°C. At the times indicated, the incubation was stopped by heating at 95°C, and the free acetate content was determined with a commercial test kit (Boehringer Mannheim). Triangles indicate the incubation of BSM with PV; squares indicate the incubation with influenza C virus.
FIG. 7
FIG. 7
(A) Cleavage of 4MUAc. PV and influenza C virus (C/JJ/50) were diluted with PBS and incubated with 0.1 mM 4MUAc for 30 min. Cleavage of substrate was monitored at a 365-nm excitation wavelength. In the first wells, 1.7 mU of PV or 0.9 mU of influenza C virus esterase was present. Reciprocals of the virus dilutions are indicated. (B) Solid-phase binding assays in microtiter plates. Microtiter wells were coated with glycoproteins (125 μg/well) or bovine brain extract (12.5 μg/well) and blocked with 3% BSA. Then 1.7 mU of PV or 0.5 mU of influenza C/JJ/50 virus was added to each well. For a control, PBS was used. After incubation for 2 h at 4°C, wells were washed three times with ice-cold PBS. Bound virus was detected by determination of the acetylesterase activity with 0.1 mM 4MUAc. The glycoconjugates used for coating are indicated on the left.

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