Abstract
BackgroundExpression of the minor virion structural protein VP2 of the calicivirus murine norovirus (MNV) is believed to occur by the unusual mechanism of termination codon-dependent reinitiation of translation. In this process, following translation of an upstream open reading frame (ORF) and termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Consistent with this, the VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 ORF (UAA) in the pentanucleotide UAA UG. Principal FindingsHere, we confirm that MNV VP2 expression is regulated by termination-reinitiation and define the mRNA sequence requirements. Efficient reintiation is dependent upon 43 nt of RNA immediately upstream of the UAA UG site. Chemical and enzymatic probing revealed that the RNA in this region is not highly structured and includes an essential stretch of bases complementary to 18S rRNA helix 26 (Motif 1). The relative position of Motif 1 with respect to the UAA UG site impacts upon the efficiency of the process. Termination-reinitiation in MNV was also found to be relatively insensitive to the initiation inhibitor edeine.ConclusionsThe termination-reinitiation signal of MNV most closely resembles that of influenza BM2. Similar to other viruses that use this strategy, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the VP1 stop codon. Our data also indicate that accurate recognition of the VP2 ORF AUG is not a pre-requisite for efficient reinitiation of translation in this system.
Highlights
For most eukaryotic mRNAs, translation initiation is a 59-enddependent process beginning with recognition of the cap structure by the cap-binding complex eIF4F [1] and recognition of the AUG codon of the first open reading frame (ORF) on the mRNA by the scanning ribosome complex [2]
We describe an analysis of termination-reinitiation in the expression of the VP2 protein of murine norovirus (MNV), a calicivirus of the genus Norovirus
The cDNA fragment was cloned in such a way that the Renilla and Firefly luciferase ORFs were in frame with the stop and start codons respectively of the termination-reinitiation motif to give an ORF configuration 59 rlucVP1-VP2fluc 39 (Figure 1)
Summary
For most eukaryotic mRNAs, translation initiation is a 59-enddependent process beginning with recognition of the cap structure by the cap-binding complex eIF4F [1] and (usually) recognition of the AUG codon of the first open reading frame (ORF) on the mRNA by the scanning ribosome complex [2]. A number of unconventional translation strategies have been described [3] These include leaky scanning of 40S subunits past the start codon of the first ORF [4], the possession of intercistronic internal ribosome entry signal [5], programmed ribosomal frameshifting during elongation [6] and stop codon suppression at the termination step [7,8]. Expression of the minor virion structural protein VP2 of the calicivirus murine norovirus (MNV) is believed to occur by the unusual mechanism of termination codon-dependent reinitiation of translation In this process, following translation of an upstream open reading frame (ORF) and termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Consistent with this, the VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 ORF (UAA) in the pentanucleotide UAAUG
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