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

Abstract

Translation of the open reading frame 2 (ORF-2) of the human respiratory syncytial virus M2 gene initiates at one of the three initiation codons located 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 demonstrated 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 translation of ORF-2 from the upstream initiation codons in vivo. This indicates that the second ORF is accessed by the ribosomes that are departing from the first ORF and that these ribosomes reinitiate on AUG codons 5' to the point of translation termination.

Fig. 1. Alignment of the sequence of the overlapping region of ORF-1 and ORF-2 of the M2 genes of pneumoviruses. Stop codons for ORF-1 are italicized and underlined. Potential initiation codons for ORF-2 are shown in bold. DDBJ/EMBL/GenBank accession numbers for the sequences shown are: human respiratory syncytial virus (HRSV) strains A2 (M74568), S2 (U39662) and B1 (AF013254); bovine respiratory syncytial virus (M82816); ovine respiratory syncytial virus (U02510); avian pneumovirus (APV, X63408); and pneumonia virus of mice (PVM; Ahmadian et al., 1999).

Fig. 2. Sequences of mutations made in the overlapping region of ORFs 1 and 2 of the RS virus M2 mRNA. The positions of start codons and mutations are shown in bold, whereas those of the stop codons are shown in bold and single underlined. The nucleotide positions of each of these in the RS virus M2 mRNA are indicated. Mutated codons are shown in bold and double underlined. M2 indicates the original RS virus strain A2 M2 mRNA sequence and Wild indicates the sequence in pWildCat. AUG-α, AUG-β and AUGs in region γ (encompassing AUGs located at positions 561–563, 567–569 and 579–581) represent potential initiation codons for the ORF-2 polypeptide.

Fig. 3. Autoradiographs showing in vitro translation protein products of recombinant plasmid constructs. Transcription and translation were directed in a cell-free coupled reaction system using T7 RNA polymerase and rabbit reticulocyte lysate. The polypeptides were analysed by SDS–PAGE on a 17% polyacrylamide gel. The positions of the molecular weight markers are indicated on the left.

Fig. 4. Diagram showing the level of expression of the CAT protein detected from recombinant plasmid constructs which had the CAT ORF fused in-frame with the second ORF of the RS virus strain A2 M2 gene. Levels of expression of CAT protein are shown as a percentage of the expression observed from plasmid pWildCat after normalization. The data are averages of the results of five experiments.

Fig. 5. Western blot analysis of polypeptides expressed in vivo. (A) Detection of M2 ORF-1 polypeptide products from the recombinant plasmid constructs pRSmut-5, pRSmut-6, pRSmut-8, pRSmut-9 and pWildCat. The lane labelled Negative is the analysis of cell lysates following transfection with plasmid pBluescribe. (B) Detection of expressed CAT protein (from ORF-2) of recombinant constructs pRSmut-1, pRSmut-2, pRSmut-8, pRSmut-9 and pWildCat.