Temperature-Sensitive Acetylesterase Activity of Haemagglutinin-Esterase Speci®Ed by Respiratory Bovine Coronaviruses
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J. Med. Microbiol. Ð Vol. 49 2000), 1119±1127 # 2000 The Pathological Society of Great Britain and Ireland ISSN 0022-2615 VIROLOGY Temperature-sensitive acetylesterase activity of haemagglutinin-esterase speci®ed by respiratory bovine coronaviruses X.Q.LIN,V.N.CHOULJENKO,K.G.KOUSOULASandJ.STORZ Department of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA Numerous respiratory bovine coronaviruses RBCV) were isolated recently from nasal swab samples and lung tissues of feedlot cattle with acute respiratory tract disease. These newly emerging RBCV isolates exhibited distinct phenotypic features that differentiated them from enteropathogenic bovine coronaviruses EBCV). The RBCV strains had a receptor-destroying enzyme function mediated by acetylesterase AE) activity of the haemagglutinin-esterase HE) glycoprotein. The HE genes of wild-type EBCV strain LY138 and RBCV strains OK-0514 OK) and LSU-94LSS-051 LSU) were cloned, sequenced and transiently expressed in COS-7 cells. The enzymic properties of HEproteins in COS-7 cellular extracts and in puri®ed virus preparations were assayed at room temperature, 378C and 398C by two different assays. One assay used rr- nitrophenyl acetate PNPA) as substrate and detected serine-esterase activity; the second assay monitored AEfunction with bovine submaxillary mucin BSM) as substrate. The PNPA tests con®rmed that HEproteins of EBCVand RBCV were functionally expressed in transfected COS-7 cells. Time-dependent determination of the AEactivity of puri®ed RBCV OK and LSU particles showed lower AEactivity at 39 8C than at 378C, whereas the puri®ed EBCV LY particles retained full AE activity at both 378C and 398C. Transiently expressed RBCV HEexhibited a marked reduction of AEactivity after 40 min of assay time at 378C. In contrast, the AEactivity of the transiently expressed EBCV HE remained stable beyond 40 min. The deduced amino-acid sequences of the HEproteins speci®ed by the RBCV strains OK and LSU contained speci®c amino-acid changes in comparison with the EBCV LY strain, which may be responsible for the observed enzymic differences. These results are consistent with the hypothesis that RBCV strains have evolved to selectively replicate in respiratory tissues and that HE may play a role in this tissue tropism. Introduction of membrane proteins [3]. It forms the short peplomer on the viral envelope [4±6]. Bovine coronavirus BCV) is a member of the Coronaviridae family and possesses a single, positive- The BCV HE serves as a receptor-binding and stranded RNA genome of 31kb in length [1,2]. receptor-destroying glycoprotein similar to the haemag- Similar to coronaviruses of antigenic group II, this glutinin-esterase-fusion HEF) glycoprotein of in¯uen- virus contains a ®fth structural protein, the haemag- za C virus [5, 7±13]. It binds to the N-acetyl-9-O- glutinin-esterase HE), in addition to the nucleocapsid acetylneuraminic acid residues of glycoproteins or N), membrane M), envelope E) and spike S) glycolipids on the surfaces of erythrocytes and proteins. This 140-kDa glycoprotein is a disulphide- susceptible cells, which is considered to be the major linked dimer of two identical 65-kDa glycosylated receptor determinant of BCV [11, 14, 15]. The recep- subunits and thus belongs to the 65-kDa class I group tor-destroying enzyme RDE) activity of BCV HE is the ®rst example of such an enzyme identi®ed on positive-stranded RNA viruses [11, 12]. Enzymes with Received 13 March 2000; revised version accepted 8 May receptor-destroying functions have been identi®ed as 2000. major structural components of negative-stranded RNA Corresponding author: Dr J. Storz. viruses such as orthomyxoviruses and paramyxoviruses 1120 X. Q. LIN ET AL. [16]. The receptor-destroying functions are mediated by of trypsin enhancement. Trypsin activation was re- neuraminidases NA) of in¯uenza A and B viruses as quired for the isolation of EBCV [32]. 2) The RBCV well as paramyxoviruses which remove terminal N- have unusually high cell-fusing activities for the G acetylneuraminate from glycoconjugates [17]. In con- clone cells. 3) The RBCV have a restricted haemag- trast, RDE of both in¯uenza C virus and BCV have glutination pattern and agglutinate only mouse and rat, acetylesterase AE) activities that hydrolyse an ester but not chicken red blood cells RBC). The EBCV linkage to release the acetyl group from position C-9 of agglutinates both rodent and chicken RBC [33]. 4) N-acetyl-9-O-acetylneuraminic acid, potentially eluting Comparative analysis of wild-type RBCV and EBCV adsorbed virions [11, 12, 14]. The 9-O-acetyl residue is nucleotide and amino-acid sequences at the 39 genomic important for in¯uenza C virus recognition of a portion 9.5 kb) revealed that RBCV-speci®c nucleotide glycoprotein receptor, a major determinant for the cell and amino-acid changes were disproportionally con- tropism [18]. centrated within the HE gene, S gene and the genomic region between the S and E genes [31]. Most of these Inhibition of the AE activity of the BCV HE peplomer RBCV isolates have RDE activities for rat RBC [34]. by diisopropyl¯uorophosphate resulted in some reduc- tion of virus infectivity, indicating that binding of HE It was hypothesised that differences in structural and to the N-acetyl-9-O-acetylneuraminic acid residue is functional properties of HE and other BCV structural involved in the infective process [11]. Enzymic proteins may cause altered tissue tropism and virus removal of the N-acetyl-9-O-acetylneuraminic acid pathogenicity. The objectives of this investigation were residue from cell membranes or treatment with HE- to identify the enzymic reaction associated with the speci®c monoclonal antibodies MAbs) inhibits BCV RDE function of HE for RBCV, to characterise the AE infections [19, 20]. Furthermore, cells infected with a activities of the HE glycoproteins from wild-type recombinant baculovirus expressing the HE of BCV strains of RBCV and EBCV in puri®ed virus prepara- exhibited haemadsorption and esterase activities, both tions and as transiently expressed gene products at of which could be blocked by MAbs with infectivity speci®c body temperatures and to compare their HE neutralisation activity [21, 22]. Consequently, it cDNA-predicted amino-acid sequences. appears that the HE of BCV is important in virus infectivity. In addition to the S glycoprotein which has strong receptor-binding properties [20, 23, 24], binding of the HE glycoprotein of the short peplomers to N- Materials and methods acetyl-9-O-acetylneuraminic acid residues on the cell Virus strains and virus puri®cation surface may function as a pre-receptor interaction for BCV. However, the HE of mouse hepatitis virus strains The wild-type strains of RBCV, OK-0514 OK) and A59 and JHM is not expressed in productive infections, LSU-94LSS-051 LSU), were used at their second to and thus is not essential for replication of these strains fourth passages. The EBCV wild-type strain LY138 [25]. LY) was used at its second and third passages. All viruses were propagated in the G clone cell line. Virus Numerous coronaviruses were isolated recently from puri®cation was performed according to Zhang et al. nasal swab samples and lung tissues of feedlot cattle [35]. Brie¯y, the media of infected G clone cell with signs of acute respiratory tract disease including a cultures were clari®ed by low speed centrifugation after severe form of shipping fever pneumonia [26±30]. three freezing and thawing cycles. Supernatant ¯uids These virus isolates multiplied only in the G clone of were collected and precipitated by polyethylene glycol human rectal tumour-18 HRT-18) cells, and not in 10% w/v. Suspensions of precipitates were loaded on to cultured bovine cells, such as Georgia bovine kidney a sucrose 20% cushion and further puri®ed by a GBK) and bovine turbinate BT) cells which are sucrose 20±60% gradient. The puri®ed virus prepara- permissive for the known respiratory bovine viruses tions were resuspended in TNE buffer 100 mM Tris- including bovine herpes virus-1 BHV-1),bovine HCl, pH 7.4, 10 mM NaCl, 1m M EDTA) and stored at parain¯uenza type-3 virus PI-3), bovine respiratory À708C. Virus infective titres in the puri®ed prepara- syncytial virus BRSV) and bovine viral diarrhoea virus tions for RBCV OK and LSU, and EBCV LY ranged BVDV). These viruses were identi®ed as respiratory from 3:0 3 109 to 4:0 3 109 plaque-forming units bovine coronaviruses RBCV) on the basis of their pfu)/ml. morphological features as determined by electron microscopy, their ability to induce cell-to-cell fusion after infection of G clone cells, and the nucleotide sequence of structural genes. Phenotypic and genotypic Assays for haemagglutinin HA) and RDE properties of RBCV differentiated them from the previously reported enteropathogenic bovine corona- The assays for HA and RDE were performed as viruses EBCV) [26±31]. Distinguishing features are as reported previously [27±29] with washed rat RBCs follows. 1) The RBCV were isolated in the ®rst G prepared as suspensions of 0.5% with PBS pH 7.4) clone cell passage at neutral pH ranges without the use containing bovine serum albumin 0.05%. HAEMAGGLUTININ-ESTERASE PROTEINS OF RBCV AND EBCV 1121 USA), counter-stained with Evans blue, and examined Cloning and sequencing of the cDNA specifying by ¯uorescence microscopy Olympus AH-2, Olympus the HE glycoprotein and alignment of predicted amino-acid sequences of different HE genes Optical, Japan). Viral RNA extraction, cDNA synthesis and DNA sequencing were performed as described previously [31], with TRI reagent Molecular Research Center, Acetylesterase assay for acetate release from Cincinnati, OH, USA), Ready-To-Go You-Prime First- bovine submaxillary mucin BSM) Strand Beads Pharmacia Biotech, Uppsala, Sweden), PCR reagents, AmpliTaq and Gene-Amp PCR system Cells in six 35-mm wells were transfected with each 9600 Perkin-Elmer, Norwalk, CT, USA), TA-cloning plasmid DNA as described above, and used to kit Invitrogen, San Diego, CA, USA), restriction determine AE activity according to Herrler et al.