Characterization of an internal ribosome entry site within mRNA 5 of murine hepatitis virus

The unique region of mRNA 5 of murine hepatitis virus contains two open rea ding frames, ORF 5a and ORF 5b. The downstream ORF 5b encodes the envelope (E) protein, an integral membrane protein of the virus. We have shown previ ously that the expression of ORF 5b is mediated by the internal entry of ri bosomes. In the experiments reported here, we have used the in vitro transl ation of synthetic mRNAs to identify the region of mRNA 5 that mediates int ernal ribosome entry. Our results show that the 5' border of the MHV mRNA 5 IRES element is located between nucleotides 227 and 244 in ORF 5a, while t he 3' border is located between nucleotides 140 and 172 in ORF 5b. The MHV mRNA 5 IRES element, therefore, contains not more than 280 nucleotides and encompasses the ORF 5b initiation codon. As evidenced by electrophoretic mo bility shift assays, the IRES element of mRNA 5 interacts specifically with protein factors present in an L-cell lysate.