Translation of the MHV sM Protein is Mediated by the Internal Entry of Ribosomes on mRNA 5
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Mouse hepatitis virus (MHV) has a positive strand RNA genome of about 31 kilobases (1). In the infected cell, viral gene expression is mediated by translation from both genomic RNA and subgenomic mRNAs. These mRNAs form a 3′ co-terminal set and they contain a common 5′leader sequence (2). Only the region of each mRNA absent from the next smallest mRNA, the so-called unique region, is thought to be translationally active (3,4). Most coronavirus mRNAs contain a single open reading frame (ORF) in their unique region and appear to be functionally mono-cistronic. One exception is the MHV mRNA 5 which contains two ORFs in its unique region, designated as ORF 5a and ORF 5b. Studies on the in vitro translation of synthetic mRNAs suggest that the MHV mRNA 5 is funtionally bicistronic (5). The ORF 5b gene product has been detected in MHV infected cells and virus particles and is equivalent to the small membrane (sM) proteins of infectious bronchitis virus (IBV) and transmissible gastroenteritis virus (TGEV)(6,7,8,9). Two mechanisms can be proposed for the expression of the MHV ORF 5b product. One possibility is based upon the leaky scanning model, as proposed by Kozak (10). In this case, the expression of ORF 5b would be mediated by ribosomes that scan from the 5′ end of the mRNA, but fail to recognise the ORF 5a initiation codon. An alternative model is a cap-independent mechanism involving ribosome entry at an internal position on the MHV mRNA 5. Such a mechanism has been described for a variety of picornavirus RNAs and hepatitis C virus RNA (11,12). In the experiments reported here, we have analysed the in vitro translation products of synthetic mRNAs that contain the unique region of MHV mRNA 5, preceded by an ORF derived from the ß-galactosidase gene. The results show that the ß-galactosidase ORF prevents the movement of ribosomes from the 5′ end of the mRNA but ORF 5b is, nevertheless, translated. We conclude that translation of the sM protein is mediated by an internal ribosome entry mechanism.
KeywordsInfectious Bronchitis Virus Translation Product Unique Region Single Open Reading Frame Mouse Hepatitis Virus