Abstract
The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.
Highlights
Reinitiation of translation is rare in eukaryotes and depends on specialized cis-acting RNA sequences tethering ribosomes to the reinitiation site
For the human Norovirus, important elements for reinitiation leading to VP2 translation are the ORF2 and ORF3 overlap region UAAUG containing the stop of the upstream ORF and the start of the downstream ORF
A first case of coupled translation termination/reinitiation relying on a novel mechanism was found in cellular mRNAs [49]
Summary
Reinitiation of translation is rare in eukaryotes and depends on specialized cis-acting RNA sequences tethering ribosomes to the reinitiation site. The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. A complementary sequence to motif 2, named motif 2*, is located directly upstream of motif 1 These two motifs were proposed to establish a secondary structure playing a role in positioning the ribosome relative to the start site of the 3Ј-terminal ORF [23, 25]. Our analyses were focused on the start site used for the reinitiation event, and we compared the results with data generated for other caliciviruses and found differences in start site recognition and common features for this interesting mechanism of translation
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