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Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry.

ABSTRACT: The internal ribosome entry sites (IRES), IRES(CP,148)(CR) and IRES(MP,75)(CR), precede the coat protein (CP) and movement protein (MP) genes of crucifer-infecting tobamovirus (crTMV), respectively. In the present work, we analyzed the activity of these elements in transgenic plants and other organisms. Comparison of the relative activities of the crTMV IRES elements and the IRES from an animal virus--encephalomyocarditis virus--in plant, yeast, and HeLa cells identified the 148-nt IRES(CP,148)(CR) as the strongest element that also displayed IRES activity across all kingdoms. Deletion analysis suggested that the polypurine (A)-rich sequences (PARSs) contained in IRES(CP,148)(CR) are responsible for these features. On the basis of those findings, we designed artificial PARS-containing elements and showed that they, too, promote internal translation from dicistronic transcripts in vitro, in tobacco protoplasts and in HeLa cells. The maximum IRES activity was obtained from multiple copies of either (A)(4)G(A)(2)(G)(2) or G(A)(2-5) as contained in IRES(CP,148)(CR). Remarkably, even homopolymeric poly(A) was moderately active, whereas a poly(G) homopolymer was not active. Furthermore, a database search for existing PARS sequences in 5'-untranslated regions (5'UTR) of genes in tobacco genome allowed the easy identification of a number of IRES candidates, in particular in the 5'UTR of the gene encoding Nicotiana tabacum heat-shock factor 1 (NtHSF1). Consistent with our prediction, the 5'UTR of NtHSF1 turned out to be an IRES element active in vitro, in plant protoplasts and HeLa cells. We predict that PARS elements, when found in other mRNAs, will show a similar activity.

SUBMITTER: Dorokhov YL

PROVIDER: S-EPMC122764 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry.

Dorokhov Yuri L YL Skulachev Maxim V MV Ivanov Peter A PA Zvereva Svetlana D SD Tjulkina Lydia G LG Merits Andres A Gleba Yuri Y YY Hohn Thomas T Atabekov Joseph G JG

Proceedings of the National Academy of Sciences of the United States of America 20020401 8

The internal ribosome entry sites (IRES), IRES(CP,148)(CR) and IRES(MP,75)(CR), precede the coat protein (CP) and movement protein (MP) genes of crucifer-infecting tobamovirus (crTMV), respectively. In the present work, we analyzed the activity of these elements in transgenic plants and other organisms. Comparison of the relative activities of the crTMV IRES elements and the IRES from an animal virus--encephalomyocarditis virus--in plant, yeast, and HeLa cells identified the 148-nt IRES(CP,148)( ...[more]

PMID: 11959981

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Project description:In recent years, hepatitis C virus (HCV)-related viruses were identified in several species, including dogs, horses, bats, and rodents. In addition, a novel virus of the genus Hepacivirus has been discovered in bovine samples and was termed bovine hepacivirus (BovHepV). Prediction of the BovHepV internal ribosome entry site (IRES) structure revealed strong similarities to the HCV IRES structure comprising domains II, IIIabcde, pseudoknot IIIf, and IV with the initiation codon AUG. Unlike HCV, only one microRNA-122 (miR-122) binding site could be identified in the BovHepV 5' nontranslated region. In this study, we analyzed the necessity of BovHepV IRES domains to initiate translation and investigated possible interactions between the IRES and core coding sequences by using a dual luciferase reporter assay. Our results suggest that such long-range interactions within the viral genome can affect IRES-driven translation. Moreover, the significance of a possible miR-122 binding to the BovHepV IRES was investigated. When analyzing translation in human Huh-7 cells with large amounts of endogenous miR-122, introduction of point mutations to the miR-122 binding site resulted in reduced translation efficiency. Similar results were observed in HeLa cells after substitution of miR-122. Nevertheless, the absence of pronounced effects in a bovine hepatocyte cell line expressing hardly any miR-122 as well suggests additional functions of this host factor in virus replication.IMPORTANCE Several members of the family Flaviviridae, including HCV, have adapted cap-independent translation strategies to overcome canonical eukaryotic translation pathways and use cis-acting RNA-elements, designated viral internal ribosome entry sites (IRES), to initiate translation. Although novel hepaciviruses have been identified in different animal species, only limited information is available on their biology on molecular level. Therefore, our aim was a fundamental analysis of BovHepV IRES functions. The findings which show that functional IRES elements are also crucial for BovHepV translation expand our knowledge on molecular mechanism of hepacivirus propagation. We also studied the possible effects of one major host factor implicated in HCV pathogenesis, miR-122. The results of mutational analyses suggested that miR-122 enhances virus translation mediated by BovHepV IRES.

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Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry.

Publications

Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry.

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