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. 2004 Feb 23;164(1):156–164. doi: 10.1016/0042-6822(88)90631-9

Detection of a murine coronavirus nonstructural protein encoded in a downstream open reading frame

Julian L Leibowitz ∗,1, Stanley Perlman †, George Weinstock ‡, James R Devries ∗, Carol Budzilowicz §, Jane M Weissemann ‡, Susan R Weiss §
PMCID: PMC7131066  PMID: 2834866

Abstract

Mouse hepatitis virus (MHV) gene 5 contains two open reading frames. We have expressed the second open reading frame of this gene (gene 5 ORF 2) in an Escherichia coli expression system. This system utilized a plasmid which contained the promoter and the first 36 codons of the recA gene fused in frame with the MHV gene 5 ORF 2, which is fused in turn to the β-galactosidase gene. The protein product of this gene fusion was used to raise antibody to gene 5 ORF 2. The specificity of the antibody was verified by immunoprecipitation of the in vitro transcribed and translated protein product of gene 5 ORF 2. The second reading frame of MHV gene 5 was shown to be expressed during the course of infection by immunocytochemistry and radioimmunoprecipitation using the antibody raised against the E. coli fusion protein and by two-dimensional gel electrophoresis.

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