Coupled Translation of the Respiratory Syncytial Virus M2 Open Reading Frames Requires Upstream Sequences

Abstract

We have investigated the mechanism of the translation of the second open reading frame (ORF) of the respiratory syncytial virus M2 transcript that uses a novel coupled translation process requiring prior translation of the upstream ORF. The second M2-2 ORF sequences play no role in the coupling process and can be replaced with other gene sequences. Surprisingly, the overlap region of the two ORFs alone was not sufficient for coupled translation to occur. An analysis of the sequences required for the coupling process showed that portions of the transcript located along the length of the first ORF M2-1, upstream of the ORF overlap region, were essential for coupled translation to occur. A critically important region for this process was centered ϳ150 nucleotides upstream of the ORF2 initiation codons. This region was shown to contain a significant degree of secondary structure, and mutation of this sequence to remove predicted areas of base pairing significantly reduced coupled translation, confirming that the secondary structure was important for the coupling process. Additional sequences further upstream increased the efficiency of the coupled translation process. These data indicate that upstream sequences act in conjunction with the M2-1/M2-2 overlap region to promote coupled translation.

A Bipartite Sequence Motif Induces Translation Reinitiation in Feline Calicivirus RNA

“…Similar results were also obtained for RHDV, where an upstream element of nearly identical length and location was necessary for translation of the 3Ј-terminal ORF (25). Influence of upstream sequences on a termination/reinitiation process was also found for the respiratory syncytial virus (RSV) M2 RNA with the two overlapping ORFs M2-1 and M2-2 (24). In RSV, the essential upstream sequence element is somewhat larger and the decrease in translation efficiency of the downstream ORF in consequence of proceeding deletions in M2-1 occurs over a longer distance, so that it is not clear at the moment whether the initiation of translation of M2-2 is mechanistically equivalent to the process described here for caliciviruses.…”

“…The biochemistry behind such a termination/reinitiation process is not fully understood. In two cases, the implication of upstream sequences has been reported although the function of these sequences is not known (24,25).…”

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination–reinitiation mechanism

“…The first ORF from each gene was amplified by PCR and the CAT reporter gene was inserted to replace the coding region of the second ORF in each gene immediately downstream from the overlap between ORF-1 and ORF-2. The entire construct was inserted into pBlueScribe as previously described (Ahmadian et al 2000;Gould and Easton 2005). This general structure of the constructs is illustrated in Figure 1A including the stop mutant.…”

“…These data also demonstrated that translation of the second ORF is not due to the presence of an IRES sequence. The region of overlap of the M2 ORF-1 and ORF-2 alone is not sufficient to achieve coupled expression but also requires additional sequences located within the M2-1 ORF (Gould and Easton 2005). Importantly, in all of these studies the data were generated in cells in which no other virus genes were being expressed, indicating that the coupled translation process is, in principle, an option available to all cells.…”

“…ORF-1 utilizes 60%-75% of the coding capacity of the mRNA and encodes the M2-1 protein product, the virus transcriptional activator (Collins and Wertz 1985;Ling et al 1992;Ahmadian et al 1999;Fearns and Collins 1999). We have demonstrated that ribosomes access and translate the second ORF in vivo in a controlled process by utilizing the three AUG codons located upstream of the ORF-1 termination codon, and expression from these initiation codons requires the prior termination of M2 ORF-1 translation (Ahmadian et al 2000;Gould and Easton 2005). The RSV M2-2 protein produced by coupled translation is thought to be involved in control of the switch between virus RNA transcription and replication (Bermingham and Collins 1999).…”