Expression of the ORF-2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism

Translation of the open reading frame 2 (ORF-2) of the human respiratory sy ncytial virus M2 gene initiates at one of the three initiation codons locat ed upstream of the termination codon for the first ORF. Replacement of ORF- 2 with the major ORF of the chloramphenicol acetyltransferase reporter gene followed by systematic mutagenesis of the putative initiation codons demon strated the usage of these codons as the translational initiators for ORF-2 expression both in vitro and in vivo. While the efficiency of translation was maintained when only the first and second AUG codons were preserved in vivo, there was no apparent preference in vitro for any of the three codons when only one was present. Mutagenesis studies showed that the location of the termination codon of ORF-1 protein plays a crucial role in directing t ranslation of ORF-2 from the upstream initiation codons in vivo. This indic ates that the second ORF is accessed by the ribosomes that are departing fr om the first ORF and that these ribosomes reinitiate on AUG codons 5' to th e point of translation termination.