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Ribosomal Frameshifting Into an Overlapping Gene in the 2B Ribosomal frameshifting into an overlapping gene in PNAS PLUS the 2B-encoding region of the cardiovirus genome Gary Loughrana,1, Andrew E. Firthb,1,2, and John F. Atkinsa,c aBioSciences Institute, University College Cork, Cork, Ireland; bDepartment of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; and cDepartment of Human Genetics, University of Utah, Salt Lake City, UT 84112-5330 AUTHOR SUMMARY −1 Proteins are essential for all −1 frameshift site acaseof frameshifting known life, and the (G GUU UUU) where the frameshift ORF sequences of their constituent 5′UTR 3′UTR is relatively short and, L1A 1B 1C 1D 2A 2B 2C 3AB 3C 3D amino acids are specified by unusually, it is completely the nucleotide sequences of StopGo 2B* transframe protein contained within the non- their template messenger frameshift ORF as an RNAs. Cellular organelles Fig. P1. Map of the EMCV genome, showing the polyprotein L-1ABCD- “overlapping gene” called ribosomes travel along 2ABC-3ABCD, the overlapping gene 2B*, and the site of programmed (Fig. P1). Cardioviruses ribosomal frameshifting. messenger RNAs, reading have an RNA genome that consecutive groups of three contains a single long ORF nucleotides called triplets, or codons, and translating each that is translated as a “polyprotein” (L-1ABCD-2ABC- triplet as an amino acid according to the “genetic code,” 3ABCD) and subsequently cleaved to produce 12 separate until they reach a “stop” codon. However, in response to proteins (3). Like many similar viruses, cardioviruses use an signals in some messenger RNAs, a proportion of ribosomes unusual ribosome-dependent mechanism, termed “StopGo” shift into a different triplet reading frame (a phenomenon or “stop-carry on,” to separate the L-1ABCD-2A segment termed programmed ribosomal frameshifting), which leads to of the polyprotein from the 2BC-3ABCD segment as the a different sequence of amino acids, and thus a different polyprotein is generated (4). Frameshifting just downstream protein. We have identified in a virus genome a previously of the 2A|2B junction results in the production of a overlooked case of ribosomal frameshifting that exhibits transframe protein termed 2B* (Fig. P1). Production of the some surprising differences from previously studied 2B* protein, and the computationally predicted site of examples. frameshifting, were verified with standard techniques Programmed ribosomal frameshifting (generally by −1 (immunoblotting, immunoprecipitation, and mass nucleotide) is used by many viruses, including HIV, SARS spectrometry) using extracts from virus-infected cells. coronavirus, and Japanese encephalitis virus. Such viruses have Mutations that abrogated expression of 2B* resulted in a BIOCHEMISTRY very compact genomes, and ribosomal frameshifting provides weakened virus that grew more slowly than the an economical mechanism for controlling gene expression and, normal virus. by establishing overlapping genes, increasing the number of Although the identification of a “hidden” gene in a virus as proteins that may be produced from a small genome. Thus, well-studied as EMCV is an interesting finding, the ribosomal frameshifting plays a crucial role in the biology and observation (below) that the frameshifting is dependent on virulence of many viruses (1). The degree to which the genes virus infection is of broader significance. To investigate the of humans and other higher organisms utilize frameshifting elements required for frameshifting in EMCV, we cloned the remains uncertain, though recent work suggests that, in shift site and surrounding sequences and placed them between addition to a few well-characterized cases, frameshifting may two genes commonly used in genetic analysis: renilla luciferase be used on a much broader scale than previously and firefly luciferase, genes used as markers because of their appreciated (2). luminescent properties. This placement made expression of Nearly all verified cases of programmed −1 frameshifting the firefly luciferase dependent on frameshifting (5). To involve two long open reading frames (ORFs, a string of calculate frameshifting efficiencies, luciferase activities were codons uninterrupted by stop codons), where frameshifting at compared to corresponding controls in which the firefly or near the end of one ORF results in translation of a luciferase is in-frame with renilla luciferase. When these following, or downstream, ORF as a “transframe” fusion with constructs of combined genes were introduced into cells, some the first ORF. Often, the two alternative protein products are of which were subsequently infected with EMCV, efficient required in a fixed ratio, and frameshifting provides an frameshifting was only observed in those cells that were economical mechanism for achieving this. Although it has infected. This result suggests that the virus produces a factor been suggested that, in certain cases, the efficiency of or modifies the cellular environment to stimulate frameshifting frameshifting may vary in a controlled fashion over the course at the EMCV frameshift site. This contrasts with previously of viral infection, there is currently little evidence for such an effect. This is in stark contrast to a handful of well- þ1 Author contributions: G.L., A.E.F., and J.F.A. designed research; G.L. and A.E.F. performed characterized cases of frameshifting (mechanistically a very research; G.L., A.E.F., and J.F.A. analyzed data; and G.L., A.E.F., and J.F.A. wrote the paper. different process) in mammals, fungi, and bacteria, where The authors declare no conflict of interest. frameshifting can play a fundamental role in the regulation of gene expression (1). This article is a PNAS Direct Submission. We performed a comparative computational analysis of Freely available online through the PNAS open access option. encephalomyocarditis virus(EMCV)sequences.EMCVisa 1G.L. and A.E.F. contributed equally to this work. member of the Cardiovirus genus, in the Picornaviridae 2To whom correspondence should be addressed. E-mail: [email protected]. family, whose genomic RNA has been very extensively used See full research article on page E1111 of www.pnas.org. to investigate translation. Using this approach, we identified Cite this Author Summary as: PNAS 10.1073/pnas.1102932108. www.pnas.org/cgi/doi/10.1073/pnas.1102932108 PNAS ∣ November 15, 2011 ∣ vol. 108 ∣ no. 46 ∣ 18579–18580 Downloaded by guest on September 30, 2021 studied −1 frameshift cases and highlights the possibility that 1. Atkins JF, Gesteland RF, eds. (2010) Recoding: Expansion of Decoding Rules Enriches the frameshifting efficiency may be modulated over the course Gene Expression (Springer, Heidelberg). of viral infection. 2. Belew AT, Advani VM, Dinman JD (2011) Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways This unusual case of frameshifting provides another in yeast. Nucleic Acids Res 39:2799–2808. example of the remarkable diversity of strategies that RNA 3. Palmenberg AC (1990) Proteolytic processing of picornaviral polyprotein. Annu Rev viruses use to control the expression of their genes. Further Microbiol 44:603–623. study of the unique features may provide new insights into the 4. Donnelly ML, et al. (2001) Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanics of protein synthesis. Moreover, the discovery mechanism indicates not a proteolytic reaction, but a novel translational effect: a highlights the potential for “hidden” frameshift ORFs within putative ribosomal ‘skip’. J Gen Virol 82:1013–1025. the genes of humans and other nonviral organisms, and the 5. Grentzmann G, Ingram JA, Kelly PJ, Gesteland RF, Atkins JF (1998) A dual-luciferase – techniques that might be employed to identify such reporter system for studying recoding signals. RNA 4:479 486. transframe genes. 18580 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1102932108 Loughran et al. Downloaded by guest on September 30, 2021.
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