Internal entry of ribosomes on a tricistronic mRNA encoded by infectious bronchitis virus.

mRNA3 specified by the coronavirus infectious bronchitis virus appears to be functionally tricistronic, having the capacity to encode three small proteins (3a, 3b, and 3c) from separate open reading frames (ORFs). The mechanism by which this can occur was investigated through in vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c ORFs, and the results suggest that translation of the most distal of the three ORFs, that for 3c, is mediated by an unconventional, cap-independent mechanism involving internal initiation. This conclusion is based on several observations. A synthetic mRNA whose peculiar 5' end structure prevents translation of the 5'-proximal ORFs (3a and 3b) directs the synthesis of 3c normally. Translation of 3c, unlike that of 3a and 3b, was insensitive to the presence of the 5' cap analog 7-methyl-GTP, and it was unaffected by alteration of the sequence contexts for initiation on the 3a and 3b ORFs. Finally, an mRNA in which the 3a/b/c infectious bronchitis virus coding region was placed downstream of the influenza A virus nucleocapsid protein gene directed the efficient synthesis of 3c as well as nucleocapsid protein, whereas initiation at 3a and 3b could not be detected. Expression of the 3c ORF from this mRNA, however, was abolished when the 3a and 3b coding region was deleted, indicating that 3c initiation is dependent on upstream sequence elements which together may serve as a ribosomal internal entry site similar to those described for picornaviruses.