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1、ELSEVIER Virus Research 35 (1995) 277-289 Virus Research Molecular characterization of the S proteins of two enterotropic murine coronavirus strains Satoshi Kunita a,1 Linong Zhang b, Felix R. Hornberger b, Susan R. Compton a,. a Section of Comparatit:e Medicine, Yale UniL,ersity School of Medicine,
2、 P.O. Box 208016, New Haven, CT 06520-8016, USA b Institute of Laboratory Animal Science, Ung, ersity of Zurich, Zurich, Switzerland Received 22 July 1994; revised 15 September 1994; accepted 15 September 1994 Abstract Enterotropic strains of routine coronaviruses (MHV-Y and MHV-RI) differ extensive
3、ly in their pathogenesis from the prototypic respiratory strains of murine coronaviruses. In an effort to determine which viral proteins might be determinants of enterotropism, im- munoblots of MHV-Y and MHV-RI virions using anti-S, -N and -M protein-specific antisera were performed. The uncleaved M
4、HV-Y and MHV-RI S proteins migrated slightly faster than the MHV-A59 S protein. The MHV-Y S protein was inefficiently cleaved. The MHV-Y, MHV-RI and MHV-A59 N and M proteins showed only minor differences in their migration. The S genes of MHV-Y and MHV-RI were cloned, sequenced and found to encode 1
5、361 and 1376 amino acid long proteins, respectively. The presence of several amino acids changes upstream from the predicted cleavage site of the MHV-Y S protein may contribute its inefficient cleavage. A high degree of homology was found between the MHV-RI and MHV-4 S proteins, whereas the homology
6、 between the MHV-Y S protein and the S proteins of other MHV strains was much lower. These results indicate that the enterotropism of MHV-RI and MHV-Y may be determined by different amino acid changes in the S protein and/or by changes in other viral proteins. Keywords: Mouse hepatitis virus; Corona
7、virus; S glycoprotein * Corresponding author. Fax: + 1 (203) 7857499. Current address: Institute of Laboratory Animal Research, School of Science, Kitasato University, Kanagawa, Japan, 0168-1702/95/$09.50 1995 Elsevier Science B.V. All rights reserved SSDI 01 68-1 702(94)00089-1 278 S. Kunita et al.
8、 / Virus Research 35 (1995) 277-289 I. Introduction Mouse hepatitis virus (MHV), a singular name for several murine coronaviruses, causes a wide spectrum of diseases ranging from mild enteritis or rhinitis to fatal hepatitis or encephalitis. MHV strains can be divided into two biotypes, respiratory
9、and enterotropic, on the basis of their initial site of replication. Following oronasal inoculation, respiratory MHV strains initiate replication in the upper respiratory tract and then disseminate to multiple organs if the mouse is sufficiently suscepti- ble due to age, genotype or immune status (B
10、arthold, 1986; Compton et al., 1993). On the other hand, replication of enterotropic MHV strains, such as MHV-RI and MHV-Y, is largely restricted to the intestinal mucosa, with minimal or no dissemi- nation to other organs (Barthold, 1987; Barthold et al., 1993). All ages and genotypes of mice are s
11、usceptible to infection with enterotropic MHV-Y but only young mice develop disease in the form of enteritis. Disease is restricted to the intestinal tract regardless of route of inoculation or immune status of the mouse (Barthold, 1987; Barthold et al., 1993). Unlike respiratory MHV strains in whic
12、h viral titers mirror the severity of lesions, the titers of enterotropic MHV produced do not reflect the level of lesion formation, in that high viral titers are produced in the intestines of MHV-Y-infected adult mice in the absence of lesions (Barthold and Smith 1987; Barthold et al., 1993). MHV v
13、irions contain two envelope glycoproteins (S and M) and an internal nucleocapsid protein (N) (Spaan et al., 1988). The S glycoprotein forms the characteristic peplomers on the virion surface. It is synthesized as a 170-200 kDa protein which is co-translationally glycosylated, oligomerized and post-t
14、ranslation- ally cleaved by trypsin-like host proteases into two subunits: the N-terminal $1 and C-terminal $2 subunits (Sturman et al., 1985). Cleavage is dependent on the virus strain and host cell type in which virus was grown (Frana et al., 1985). The S protein is believed to be responsible for
15、the initiation and spread of infection, by mediating the attachment of the virus to cell surface receptors and by cell to cell fusion (Collins et al., 1982; Dveksler et al., 1991; Williams et al., 1991). The S protein also elicits neutralizing antibodies and cellular immune responses (Collins et al.
16、, 1982; Wege and Dorries, 1984; Korner et al., 1991; Mobley et al., 1992). The M protein is a 20-30 kDa integral membrane glycoprotein. It accumulates in the membranes of the Golgi apparatus where it interacts with viral nucleocapsids and determines the site of virus budding (Armstrong et al., 1984; Tooze et al., 1984). The N protein is a 43-60 kDa phosphoprotein which binds to viral genomes to form helical nucleocapsids (Stohlman and Lai, 1979). The molecular basis of MHV pathoge
