Expression of the VP2 Protein of Murine Norovirus by a Translation Termination-Reinitiation Strategy

Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK.
PLoS ONE (Impact Factor: 3.23). 12/2009; 4(12):e8390. DOI: 10.1371/journal.pone.0008390
Source: PubMed


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.
Here, 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 UAAUG 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 UAAUG site impacts upon the efficiency of the process. Termination-reinitiation in MNV was also found to be relatively insensitive to the initiation inhibitor edeine.
The 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.

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Available from: Richard J Jackson, Aug 14, 2014
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    • "That the predicted pseudoknot must be located closely upstream of the CP stop codon (Li et al., 2011) suggests a functional role for this structure in tethering the terminating ribosome or components thereof, which can then reinitiate with limited efficiency at a nearby start codon. These findings and suggested mechanism for victoriviruses are reminiscent of translational reinitiation as characterized to date in influenza B viruses and caliciviruses, which contain a required RNA region termed the TURBS within 90 nt upstream of the stop/restart codons (Horvath et al., 1990; Luttermann and Meyers, 2007, 2009, 2014; McCormick et al., 2008; Meyers, 2003, 2007; Napthine et al., 2009; Powell et al., 2008, 2011; Pöyry et al., 2007). The TURBS functions in part via a short sequence within it that base-pairs with host 18S rRNA and thereby helps to retain the 40S ribosome after translational termination on the upstream ORF. "
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    ABSTRACT: Prototype victorivirus HvV190S employs stop/restart translation to express its RdRp from the downstream ORF in its bicistronic mRNA. The signals for this activity appear to include a predicted RNA pseudoknot directly upstream of the CP stop and RdRp start codons, which overlap in the motif AUGA. Here we used a dual-fluorescence system to further define which HvV190S sequences are important for stop/restart translation and found that the AUGA motif plus 38 nt directly upstream are both necessary and sufficient for this activity. This RNA cassette encompasses the predicted pseudoknot, and indeed substitutions that disrupted the pseudoknot disrupted the activity whereas complementary substitutions that restored the pseudoknot restored the activity. Replacement of this RNA cassette with those from other victoriviruses with a predicted pseudoknot in comparable position also supported stop/restart translation. To our knowledge, this is the first example of stop/restart translation regulated by an RNA pseudoknot.
    Virology 11/2014; 474. DOI:10.1016/j.virol.2014.10.022 · 3.32 Impact Factor
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    • "Taxon Products References Caliciviridae – Lagovirus, Vesivirus, Norovirus, Sapovirus, Nebovirus VP1/VP2 Meyers (2003); Luttermann & Meyers (2007); Napthine et al. (2009) "
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    ABSTRACT: Viral protein synthesis is completely dependent upon the translational machinery of the host cell. However, many RNA virus transcripts have marked structural differences from cellular mRNAs that preclude canonical translation initiation, such as the absence of a 5' cap structure or the presence of highly structured 5'UTRs containing replication and/or packaging signals. Furthermore, whilst the great majority of cellular mRNAs are apparently monocistronic, RNA viruses must often express multiple proteins from their mRNAs. In addition, RNA viruses have very compact genomes and are under intense selective pressure to optimize usage of the available sequence space. Together, these features have driven the evolution of a plethora of non-canonical translational mechanisms in RNA viruses that help them to meet these challenges. Here, we review the mechanisms utilized by RNA viruses of eukaryotes, focusing on internal ribosome entry, leaky scanning, non-AUG initiation, ribosome shunting, reinitiation, ribosomal frameshifting and stop-codon readthrough. The review will highlight recently discovered examples of unusual translational strategies, besides revisiting some classical cases.
    Journal of General Virology 04/2012; 93(Pt 7):1385-409. DOI:10.1099/vir.0.042499-0 · 3.18 Impact Factor
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    • "The paucity of stable RNA secondary structure within the BM2 TURBS [11], the TURBS of FCV [I. Brierley, unpublished observations] and the calicivirus murine norovirus (MNV; [20]), limits the predictive power of RNA folding programs in the assessment of the likely RNA folds present before and after ribosomal transit through the TURBS. It is also known that eukaryotic initiation factor 3 (eIF3) plays a role in the termination-reinitiation process, perhaps interacting with the TURBS to provide another link between mRNA and the 40S subunit [17]. "
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    ABSTRACT: Termination-dependent reinitiation is used to co-ordinately regulate expression of the M1 and BM2 open-reading frames (ORFs) of the dicistronic influenza B segment 7 RNA. The start codon of the BM2 ORF overlaps the stop codon of the M1 ORF in the pentanucleotide UAAUG and ∼10% of ribosomes terminating at the M1 stop codon reinitiate translation at the overlapping AUG. BM2 synthesis requires the presence of, and translation through, 45 nt of RNA immediately upstream of the UAAUG, known as the 'termination upstream ribosome binding site' (TURBS). This region may tether ribosomal 40S subunits to the mRNA following termination and a short region of the TURBS, motif 1, with complementarity to helix 26 of 18S rRNA has been implicated in this process. Here, we provide further evidence for a direct interaction between mRNA and rRNA using antisense oligonucleotide targeting and functional analysis in yeast cells. The TURBS also binds initiation factor eIF3 and we show here that this protein stimulates reinitiation from both wild-type and defective TURBS when added exogenously, perhaps by stabilising ribosome-mRNA interactions. Further, we show that the position of the TURBS with respect to the UAAUG overlap is crucial, and that termination too far downstream of the 18S complementary sequence inhibits the process, probably due to reduced 40S tethering. However, in reporter mRNAs where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. Reinitiation on distant AUGs is not inhibited in eIF4G-depleted RRL, suggesting that the tethered 40S subunit can move some distance without a requirement for linear scanning.
    PLoS ONE 02/2011; 6(2):e16822. DOI:10.1371/journal.pone.0016822 · 3.23 Impact Factor
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