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      Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production

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          Abstract

          The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 3′-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 5′ end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43–64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection.

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          Most cited references46

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          The molecular biology of arteriviruses.

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            Pushing the limits of the scanning mechanism for initiation of translation.

            Selection of the translational initiation site in most eukaryotic mRNAs appears to occur via a scanning mechanism which predicts that proximity to the 5' end plays a dominant role in identifying the start codon. This "position effect" is seen in cases where a mutation creates an AUG codon upstream from the normal start site and translation shifts to the upstream site. The position effect is evident also in cases where a silent internal AUG codon is activated upon being relocated closer to the 5' end. Two mechanisms for escaping the first-AUG rule--reinitiation and context-dependent leaky scanning--enable downstream AUG codons to be accessed in some mRNAs. Although these mechanisms are not new, many new examples of their use have emerged. Via these escape pathways, the scanning mechanism operates even in extreme cases, such as a plant virus mRNA in which translation initiates from three start sites over a distance of 900 nt. This depends on careful structural arrangements, however, which are rarely present in cellular mRNAs. Understanding the rules for initiation of translation enables understanding of human diseases in which the expression of a critical gene is reduced by mutations that add upstream AUG codons or change the context around the AUG(START) codon. The opposite problem occurs in the case of hereditary thrombocythemia: translational efficiency is increased by mutations that remove or restructure a small upstream open reading frame in thrombopoietin mRNA, and the resulting overproduction of the cytokine causes the disease. This and other examples support the idea that 5' leader sequences are sometimes structured deliberately in a way that constrains scanning in order to prevent harmful overproduction of potent regulatory proteins. The accumulated evidence reveals how the scanning mechanism dictates the pattern of transcription--forcing production of monocistronic mRNAs--and the pattern of translation of eukaryotic cellular and viral genes.
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              Nidovirus transcription: how to make sense...?

              Many positive-stranded RNA viruses use subgenomic mRNAs to express part of their genetic information. To produce structural and accessory proteins, members of the order Nidovirales (corona-, toro-, arteri- and roniviruses) generate a 3' co-terminal nested set of at least three and often seven to nine mRNAs. Coronavirus and arterivirus subgenomic transcripts are not only 3' co-terminal but also contain a common 5' leader sequence, which is derived from the genomic 5' end. Their synthesis involves a process of discontinuous RNA synthesis that resembles similarity-assisted RNA recombination. Most models proposed over the past 25 years assume co-transcriptional fusion of subgenomic RNA leader and body sequences, but there has been controversy over the question of whether this occurs during plus- or minus-strand synthesis. In the latter model, which has now gained considerable support, subgenomic mRNA synthesis takes place from a complementary set of subgenome-size minus-strand RNAs, produced by discontinuous minus-strand synthesis. Sense-antisense base-pairing interactions between short conserved sequences play a key regulatory role in this process. In view of the presumed common ancestry of nidoviruses, the recent finding that ronivirus and torovirus mRNAs do not contain a common 5' leader sequence is surprising. Apparently, major mechanistic differences must exist between nidoviruses, which raises questions about the functions of the common leader sequence and nidovirus transcriptase proteins and the evolution of nidovirus transcription. In this review, nidovirus transcription mechanisms are compared, the experimental systems used are critically assessed and, in particular, the impact of recently developed reverse genetic systems is discussed.
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                Author and article information

                Journal
                J Gen Virol
                JGV
                vir
                The Journal of General Virology
                Society for General Microbiology
                0022-1317
                1465-2099
                May 2011
                : 92
                : Pt 5
                : 1097-1106
                Affiliations
                [1 ]Department of Pathology, University of Cambridge, Cambridge, UK
                [2 ]Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
                [3 ]Department of Human Genetics, University of Utah, Salt Lake City, UT 84112-5330, USA
                [4 ]Department of Veterinary Science, Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099, USA
                [5 ]BioSciences Institute, University College Cork, Cork, Ireland
                Author notes
                Correspondence Eric J. Snijder e.j.snijder@ 123456lumc.nl Clara C. Posthuma c.c.posthuma@ 123456lumc.nl
                Article
                029264
                10.1099/vir.0.029264-0
                3139419
                21307223
                7f07f426-7da5-4fd6-a66d-21a884c54475
                Copyright @ 2011

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 November 2010
                : 07 February 2011
                Funding
                Funded by: Wellcome Trust
                Award ID: 088789
                Funded by: Science Foundation Ireland gran
                Award ID: 08/IN.1/B1889
                Funded by: National Institutes of Health grant
                Award ID: R01 GM079523
                Categories
                Standard
                Animal
                RNA
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                David Eyre
                Marianne Asbury
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                Microbiology & Virology
                Microbiology & Virology

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