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 approximately 150 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.
Highlights
It has long been recognized that mRNA is a central molecule in the parasitic interaction of a virus with its host [1]
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
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
Summary
Plasmid Construction—All of the PCR reactions were carried out using PfuTurbo Hotstart DNA polymerase (Stratagene, Amsterdam, The Netherlands), and resulting constructs were sequenced. Primers C28 and pBS-SphImut were used to amplify the M2 overlap sequence and the CAT ORF (cloned in-frame with the M2-2 start codons in the M2 overlap region) from pWildCAT (Fig. 2A) This PCR product was digested with SphI and HindIII and ligated along with the digested EGFP PCR product into pBS digested with KpnI and HindIII to generate pC28 (Fig. 1D). When using pWildCAT as the template with the M2-1-specific primer and pBS-SphImut, a designated portion of M2-1 and the CAT ORF was amplified (Fig. 2A) These various sized M2-1-CAT products were digested with SphI and HindIII and cloned into the 3917-bp fragment so that they could be positioned downstream of EGFP.
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