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

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 beta-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.

Internal Ribosome Entry in the Coding Region of Murine Hepatitis Virus mRNA 5

“…2a, lane 5). This result is consistent with the idea that the M H V m R N A 5 is functionally bicistronic (Skinner et al, 1984;Budzilowicz & Weiss, 1987;Leibowitz et al, 1988). In the experiment shown, the ratio of O R F 5a to O R F 5b products translated from m R N A 5ab was about 8:1, showing that, at least in this system, O R F 5a is more efficiently translated than O R F 5b.…”

“…MHV mRNA 5 also contains two ORFs in its unique region, designated ORF 5a and ORF 5b. In vitro translation studies suggest that this mRNA is functionally bicistronic and the ORF 5b gene product has been detected in MHV (strain A59)-infected cells Leibowitz et al, 1988). On the basis of sequence similarities, it is likely that the MHV ORF 5b gene product is equivalent to the small membrane (sM) proteins of infectious bronchitis virus (IBV) and transmissible gastroenteritis virus particles (Liu & Inglis, 1991;Godet et al, 1992).…”

Identification of a New Membrane-associated Polypeptide Specified by the Coronavirus Infectious Bronchitis Virus

“…Among the other coronaviruses, there is also some suggestion that subgenomic mRNAs are functionally polycistronic. Nucleotide sequencing of both the A59 and JHM strains of murine hepatitis virus (MHV) indicates that mRNA 5 contains two overlapping ORFs Budzilowicz & Weiss, 1987), and a 9-6K product from the more distal of these has been detected in infected cells (Leibowitz et al, 1988).…”

Coronaviruses: Structure and Genome Expression

“…In vitro translation of RNA transcribed from cDNA containing both ORFs of MHV-A59 mRNA 5 indicated that the downstream reading frame is preferentially translated (Budzilowicz & Weiss, 1987). This ORF was shown to be expressed in infected cells (Leibowitz et al, 1988).…”