Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA

Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
RNA (Impact Factor: 4.94). 12/2011; 18(2):241-52. DOI: 10.1261/rna.030338.111
Source: PubMed


Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.

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    • "Although the mechanism of inducing −1 FS is distinct from +1 FS or stop codon RT, a common feature of these recoding events is the involvement of a 3′ stimulatory RNA structure (40,41). To investigate if RNA G4 can induce +1 FS or stop codon RT, we replaced the well-characterized +1 FS stimulatory pseudoknot of mammalian antizyme (42) and the RT stimulatory stem-loop structure of Colorado tick fever virus segment 9 (43) with the most stable (G3U)4 G4 sequence (Figure 5). Interestingly, the RNA G4 could induce significant levels of +1 FS (3.0%) and stop codon RT (1.5%) against a background of 0.6 and 0.3%, respectively (Figure 5). "
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    ABSTRACT: Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating -1 ribosomal frameshifting (-1 FS) in vitro and in cultured cells. Systematic manipulation of the loop length between each G-tract revealed that the -1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher -1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.
    Nucleic Acids Research 10/2013; 42(3). DOI:10.1093/nar/gkt1022 · 9.11 Impact Factor
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    • "Pomovirus RNA2 CP-extension I UAG-CAA-UYA, UAA-CAA-UUA Luteovirus, Polerovirus CP-extension III AAA-UAG-GUA Long-distance 39 base pairing? Brown et al. (1996); Bruyère et al. (1997) Rose spring dwarfassociated luteovirus CP-extension II UGA-CGG Enamovirus CP-extension UGA-GGG Demler & de Zoeten (1991) Benyvirus CP-extension I UAG-CAA-UUA Compact stem–loop* Rice stripe necrosis benyvirus CP-extension III UAG-GGG Compact stem–loop* Coltivirus, segment 9 VP99 II UGA-CGG Extended stem–loop Jaafar et al. (2004); Napthine et al. (2012) "
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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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    ABSTRACT: The rotavirus (RV) genome consists of eleven segments of double-stranded (ds)RNA. Typically, each segment contains 5' and 3' untranslated regions (UTRs) that flank an open reading frame (ORF) encoding a single protein. RV variants have been described with segments of atypical size owing to sequence rearrangements. In many cases, the rearrangement originates from a partial head-to-tail sequence duplication that initiates after the stop codon of the ORF, leaving the protein product of the segment unaffected. To probe the limits of the RV genome to accommodate additional genetic sequence, we used reverse genetics to insert duplications (analogous to synthetic rearrangements) and heterologous sequences into the 3' UTR of the segment encoding NSP2 (gene 8). The approach allowed the recovery of recombinant RVs that contained sequence duplications (up to 200 bp) and heterologous sequences including those for FLAG, the hepatitis C virus E2 epitope, and the internal ribosome entry site of cricket paralysis virus. The recombinant RVs grew to high titer (>10(7) PFU/ml) and remained genetically stable during serial passage. Despite their longer 3' UTRs, rearranged RNAs of recombinant RVs expressed wildtype levels of protein in vivo. Competitive growth experiments indicated that, unlike RV segments with naturally occurring sequence duplications, genetically engineered segments were packaged less efficiently into progeny viruses. Thus, features of naturally occurring rearranged segments, other than their increased length, contribute to their enhanced packaging phenotype. Our results define strategies for developing recombinant RVs as expression vectors, potentially leading to next-generation RV vaccines that induce protection against other infectious agents.
    Journal of Virology 03/2013; 87(11). DOI:10.1128/JVI.00413-13 · 4.44 Impact Factor
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