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1、Characterization of the expression and immunogenicity of the ns4b protein of human coronavirus 229E Fanny Chagnon, Alain Lamarre, Claude Lachance, Michelle Krakowski, Trevor Owens, Jean-Franois Lalibert, and Pierre J. Talbot Abstract: Sequencing of complementary DNAs prepared from various coronaviru
2、ses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression and immunogenicity of the ns4b gene product from strain 229E of hu
3、man coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used to demonstrate the expression of ns4b in HCV-229
4、E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice to evaluate whether myelinviral protein
5、cross-reactive antibody responses could be generated. Each immunogen induced specific but not cross-reactive antibodies. We conclude that ns4b is expressed in infected cells and is immunogenic, although this does not involve amino acids shared with a self protein, at least in the experimental condit
6、ions used. Key words: human coronavirus 229E, nonstructural protein, ns4b protein, expression, immunogenicity. Rsum : Le squenage dADN complmentaires prpars partir de divers coronavirus a rvl des cadres de lecture ouverts codant dhypothtiques protines qui ne sont pas encore caractrises et que lon no
7、mme protines non structurales (ns). Dans une premire tape de la caractrisation de sa fonction, nous avons tudi lexpression et limmunognicit du produit du gne ns4b de la souche 229E du coronavirus humain (HCV-229E), un virus respiratoire possdant un potentiel neurotrope. Le gne a t clon et exprim dan
8、s des bactries. Une protine de fusion de ns4b avec la protine liant le maltose a t injecte des lapins afin de produire un antisrum spcifique qui a ensuite t utilis pour dmontrer, par cytomtrie de flux, lexpression de ns4b dans des cellules infectes. tant donn notre observation antrieure dune squence
9、 de cinq acides amins partage entre ns4b et la protine basique de la myline, une protine ns4b recombinante comprenant une queue de rsidus histidine et (ou) la protine basique de la myline humaine ont t injectes des souris pour valuer linduction possible danticorps montrant une raction croise envers
10、ces deux protines. Chaque immunogne a induit des anticorps spcifiques qui ne prsentaient pas de ractions croises. Nous concluons que la protine ns4b est exprime dans les cellules infectes et quelle est immunogne, quoique cette rponse immunitaire ne ciblait pas les acides amins partags avec une proti
11、ne du soi, au moins dans les conditions exprimentales utilises. Mots cls : coronavirus humain 229E, protine non structurale, protine ns4b, expression, immunognicit. Notes 1017 Human coronaviruses (HCV) are known to cause between 15 and 35% of common colds (McIntosh 1974; Myint 1994). Coronaviruses p
12、ossess a single-stranded, positive- sense RNA genome of more than 30 kb (Holmes and Lai 1996). In infected cells, six subgenomic RNAs constitute a nested set of 3-coterminal mRNA species, of which only the 5-unique region appears to be translated (Holmes and Lai 1996). Of those mRNAs, four encode st
13、ructural proteins found in the virion: in HCV-229E infected cells, they are the 50- to 60-kDa nucleocapsid N protein associated with genomic RNA (Schreiber et al. 1989); the 21- to 25-kDa M glycoprotein associated with the viral envelope (Jouvenne et al. 1990; Raabe and Siddell 1989a); the 170- to 2
14、00-kDa S glycoprotein that forms viral spikes (Raabe et al. 1990); and the 9- to 12-kDa E protein, a small membrane protein with unknown functions (Holmes and Lai 1996). In addition to Can. J. Microbiol. 44: 10121017 (1998) 1998 NRC Canada 1012 Received February 19, 1998. Revision received July 6, 1
15、998. Accepted August 5, 1998. F. Chagnon, A. Lamarre, C. Lachance, and P.J. Talbot.1 Laboratory of Neuroimmunovirology and Human Health Research Center, Institut Armand-Frappier, INRS, Universit du Qubec, 531, boulevard des Prairies, Laval, QC H7V 1B7, Canada. M. Krakowski and T. Owens. Neuroimmunol
16、ogy Unit, Montreal Neurological Institute, McGill University, 3801 University, Montral, QC H3A 2B4, Canada. J.-F. Lalibert. Microbiology and Biotechnology Research Center, Institut Armand-Frappier, INRS, Universit du Qubec, 531, boulevard des Prairies, Laval, QC H7V 1B7, Canada. 1Author to whom all correspondence should be sent at the following address: Laboratoire de neuroimmunovirologie, Institut Armand-Frappier, 531, boulevard des Prairies, Laval, QC H7V 1B7, Canada (e-mail: Pierre.Talboti
