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1、VIROLOGY 189, 792-794 (1992) Neuraminidase Treatment of Avian Infectious Bronchitis Coronavirus Reveals a Hemagglutinating Activity That is Dependent on Sialic Acid-Containing Receptors on Erythrocytes BEATE SCHULTZE,*. DAVID CAVANAGH,t AND GEORG HERRLER* *Institut fiir virologie, Phi/tops-Universit
2、;it Marburg, Robert-Koch-Str. 17, 3550 #arburg, Germany; and iAgricultura/ and Food Research Council, Institute for Animal Health, Houghton Laboratory, Houghton, Huntingdon PE 17 2DA, UK Received February 14, 1992; accepted April 16, 1992 The interaction of infectious bronchitis virus (IBV) with ery
3、throcytes was analyzed. The binding activity of IBV was not sufficient to agglutinate chicken erythrocytes. However, it acquired hemagglutinating activity after treatment with neuraminidase to remove 200 mu/ml) or Newcastle disease virus (NDV; 3.5 U/ml), IBV was purified by sucrose gradient centrifu
4、gation (4). The final viral pellet was suspended In 100 pl of PBS and used for hemagglutlnation assays with chicken erythro- cytes (6). The bacterial neuraminldase was purchased from Behr- Ing-Werke (Marburg, Germany). The viral enzyme was isolated by detergent (octylglucoside) treatment of purified
5、 egg-grown NDV fol- lowed by sucrose gradient centrifugation (4). Fractions containing neuraminidase were collected and dialyzed to remove sucrose and detergent. cial component of the erythrocyte receptors for IBV. To confirm this finding, neuraminidase-treated cells were resialylated by incubation
6、with GalPI ,3GalNAc cu2,3sialyltransferase, and CMP-Neu5Ac. Following at- tachment of sialic acid to the surface of erythrocytes in an c2,3 linkage, the ceils were agglutinated by both NDV and IBV (Table 3). The former virus is known to recognize cu2,3-linked sialic acid as a receptor determi- nant
7、for attachment to cells (1 I). Our results indicated that IBV had the same requirements for agglutination of erythrocytes as NDV. All viruses that have been reported so far to use sia- lit acid for attachment to cells have found a way to keep the viral surface free of the receptor determinant. TABLE
8、 2 INACTIVATION OF ERYTHROCYTE RECEPTORS FOR IBV BY NEURAMINIDASE Pretreatment of ceils Hemagglutinating activity of neuraminldase-treated IBV (HA units/ml) None 512 VC-neuramtnldase 4 NDV-neuraminidase 2 Note. Erythrocytes from 1 -day-old chickens (300 1, 10% suspen- slon In PBS) were incubated In
9、the absence or presence of neuramlni- dase from Vibrio cholerae (VC, 23 mu) or Newcastle disease virus (NDV, 350 mu) for 90 min at 37C. Cells were washed and used to determine the hemagglutination titer of IBV which had been pre- treated with neuraminidase from NDV to induce the hemagglutinat- Ing a
10、ctivity (see Table 1). Erythrocytes IBV NDV Control Asialo Resialylated, Gala2,3Neu5Ac 512 256 t2 2 256 64 Note. A 10% suspension of chicken erythrocytes was Incubated with neuramlnidase from Vibrio cholerae (40 mu/ml) for 30 min at 37C. Asialo cells were washed and suspended in PBS to a final conce
11、ntration of 27.5% in a total volume of 52 1. Resialylation was accomplished by incubation with sialyltransferase (8 mU; from Boehringer-Mannhelm, Germany) and 250 nmol of CMP-activated N-acetylneuraminic acid (Neu5Ac). After 2 hr at 37”C, cells were washed and used as a 1% suspension (in PBS) to det
12、ermine the hemagglutinatlng activity of IBV and Newcastle disease virus (NDV). The HA activity of IBV had been induced by pretreatment with NDV- neuraminldase (see Table 1). In order to protect the cells from lysls by the detergent present in the sialyltransferase preparation, fixed erythrocytes wer
13、e used for this experiment (0.1% glutaraldehyde, 60 min). Reoviruses, polyomavirus, and encephalomyocarditis virus are nonenveloped viruses without glycoconju- gates containing sialic acid. Enveloped viruses contain both glycoproteins and glycolipids. However, some of these viruses possess a recepto
14、r-destroying enzyme, which is responsible for the lack of sialic acid on the surface (10): a neuraminidase in the case of influenza A and B viruses and paramyxoviruses, an acetylesterase in the case of influenza C virus and several coronavi- ruses. IBV is the first enveloped virus reported to recog-
15、 nize c-u2,3-linked sialic acid which lacks a receptor-des- troying enzyme. There are several ways to explain the masking effect of sialic acid on the hemagglutinating activity of IBV: (i) The inhibitory sialic acid of a virion might interact with the viral binding protein of another virus particle,
16、 result- ing in the formation of virus aggregates; (ii) The sialic acid and the viral attachment protein may be part of the same virion, e.g., the interaction between neighboring S-proteins; (iii) The sialic acid molecule may be part of a cellular component, which is bound by the virion and acts as an inhibitor of the viral hemagglutinating activ- ity. The first possibility can be excluded because there is no indication by electron
