Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

Laboratory of Hepatitis and Related Emerging Agents, Division of Emerging and Transfusion-Transmitted Diseases, Office of Blood Research and Review, CBER, FDA, 8800 Rockville Pike, HFM310, Bethesda, Maryland 20892, USA.
Journal of Virology (Impact Factor: 4.65). 02/2010; 84(9):4330-40. DOI: 10.1128/JVI.02480-09
Source: PubMed

ABSTRACT In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

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    ABSTRACT: Programmed -1 ribosomal frameshifting (-1RFS) is one of well-known alternative decoding mechanisms found in nature. -1RFS mechanism has been first described as a mechanism that controls the relative expression levels of two proteins in metazoan viruses. 1 Furthermore, among many retroviruses, plant viruses, coronaviruses and certain bacterial and protozoan genes, this has been identified as a mechanism that modulates the translation of two proteins encoded by overlapping open reading frames present in one mRNA. 2,3 Their prevalence across such an evolutionarily diverse distribution manifests that such sites have evolved several times. Sequence comparison and molecular genetic analysis of many of -1RFS sites show a canonical structure for these frameshift sites. It is now clear that two cis-elements in mRNA alone are enough to create this unusual alternative decoding event, although there are some possible influences of other trans-factors on frameshifting efficiency. Two cis-elements are involved in this alternative reading process of genetic codes during translation. 4 One is the slippery sequence, a heptanucleotide motif XXXYYYN, where -1 frameshifting occurs. The other is a downstream RNA secondary structure, usually a pseudoknot. The -1RFS system containing these two cis-elements can induce -1 frameshift-ing at a slippery site (slippery sequence) with 1 to over 30% of efficiency. Recent studies have worked on not only the detailed mechanism and functional importance of -1RFS, 5-7 but also possible utilization of -1RFS for regulation of gene expression. For example, Dinman's group demonstrated that -1RFS signal from L-A virus successfully functions as a cis-acting mRNA destabilizing element. 8 In addition, small ligands targeting the embedded biotin-binding RNA sequence on mRNA that forms a pseudoknot-like structure when it bound with a biotin is sufficient for stimulating -1RFS when they replace biotin to bind the pseudoknot. Here, we carried out the experiments to place -1RFS signal into genes of interest. This study was aimed to answer two questions. Firstly, can efficient -1RFS occur after very preceding translation, e.g. a less than 10 amino acid residues? Secondly, what is the effect of the preceding translation length on the efficiency of -1RFS? We used the following experimental strategy. As a reliable assay system for the functional analysis of -1RFS in vivo, we chose the dual-luciferase assay system that measures the luminescence activities of Renilla luciferase (Rluc) and firefly luciferase (fluc) by a luminometer. In this assay system, activities of two luciferase-gene products are subsequently measured in the same tube. Thus, relative amounts of two luciferase proteins that reflect efficiency of -1RFS events can be determined by the activities of two luciferases. The reporter vectors were designed to have both Rluc and fluc genes in two separate open reading frames in the oppo-site direction using pBI-L (BD Biosciences) vector as a Figure 1. Schematic map of the pBI-2lucL reporter vector and the reporter gene expression by -1RFS. (a) Map of the pBI-2lucL reporter vector. The reporter vector, pBI-2lucL, has two luciferase genes, firefly luciferase (fluc) and Renilla luciferase (Rluc), that are oriented in the opposite directions but both under the control of the same bidirectional promoter, Pbi-1. The -1RFS signal (FS) is placed between an ORF1 and the 5' region of the Rluc gene that fused in -1 frame. Three genes with different lengths were fused to the Rluc gene via the FS; genes for the Zα domain from human ADAR1 (Zα), thioredoxin protein (Trx) and glutathione S-transferase protein (GST). (b) Fusion protein products from various ORF1-reporter gene constructs by -1RFS. ORF1-Rluc fusion proteins are produced only when -1RFS occurs at the FS. Fusion proteins produced from -1RFS event were detected by an anti-Renilla luciferase monoclonal antibody (left panel).
    Bulletin- Korean Chemical Society 09/2011; 32(9). DOI:10.5012/bkcs.2011.32.9.3489 · 0.84 Impact Factor
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    ABSTRACT: Translational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial. IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.
    Journal of Virology 07/2014; 88(18). DOI:10.1128/JVI.00898-14 · 4.65 Impact Factor
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    ABSTRACT: Programmed -1 ribosomal frameshifting (-1 PRF) stimulated by mRNA pseudoknots regulates gene expression in many viruses, making pseudoknots potential targets for anti-viral drugs. The mechanism by which pseudoknots trigger -1 PRF, however, remains controversial, with several competing models. Recent work showed that high -1 PRF efficiency was linked to high pseudoknot conformational plasticity via the formation of alternate conformers. We tested whether pseudoknots bound with an anti-frameshifting ligand exhibited a similar correlation between conformational plasticity and -1 PRF efficiency, by measuring the effects of a ligand which was found to inhibit -1 PRF in the SARS coronavirus on the conformational dynamics of the SARS pseudoknot. Using single-molecule force spectroscopy to unfold pseudoknots mechanically, we found that the ligand binding effectively abolished the formation of alternate conformers. This result extends the connection between -1 PRF and conformational dynamics, and moreover suggests that targeting the conformational dynamics of pseudoknots may be an effective strategy for anti-viral drug design.
    Journal of the American Chemical Society 01/2014; 136(6). DOI:10.1021/ja410344b · 11.44 Impact Factor


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