A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5' UTR

Dumas, Estelle; Staedel, Cathy; Colombat, Marie; Reigadas, Sandrine; Chabas, Sandrine; Astier-Gin, T.; Cahour, Annie; Litvak, Simón; Ventura, Michel

doi:10.1093/nar/gkg199

Cited by 47 publications

(48 citation statements)

References 22 publications

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“…In our study, translation from defective HCV IRES mutants remained higher than the MCS control. We expect that these residual activities are most likely explained by cryptic promoter activity in the HCV IRES sequence (31), and/or by other interactions between the IRES and 40S subunits that are capable of supporting low levels of expression. To assess the contribution of cryptic promoter activity in our system, we tested a promoterless construct (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Matsuda

Mauro

2014

Proc. Natl. Acad. Sci. U.S.A.

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Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example-translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson-Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA-rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the ShineDalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.hepatitis C virus | 18S rRNA | IRES | base pairing | translation H CV is a single-stranded RNA virus that is a major cause of severe liver disease. The RNA genome contains a large ORF and expresses a single polypeptide that is processed into smaller proteins, which are necessary for replication and assembly of viral particles. The RNA genome is uncapped, and translation does not require the eukaryotic initiation factor 4F (eIF4F) complex (1), which mediates cap-dependent translation. Instead, translation is facilitated by an internal ribosome entry site (IRES) located in the 5′ nontranslated region (2, 3). Inasmuch as the production of all HCV proteins requires the HCV IRES, the HCV IRES is a therapeutic target (4).IRESs encompass a variety of initiation mechanisms and have been extensively studied in viruses, which often exploit the translation capabilities of the host for their own use (5-7). In many cases, the IRES mechanism complements the infection strategy of the virus. In the case of the HCV IRES and other IRESs of this type, the IRES can effectively recruit the host translation machinery by directly binding to 40S ribosomal subunits (1,(8)(9)(10)(11)(12). The HCV-ribosome interaction has been shown to require an interaction with ribosomal protein S25 (13) and can also involve the eIF3 complex, which increases the stability of the interaction (14). In the presence of ternary complex, which contains the initiator Met-tRNA, a preinitiation complex can assemble at the start site.Various studies have investigated the binding of ...

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Section: Resultsmentioning

confidence: 99%

“…Finally, to further address potential issues associated with IRES analyses using DNA reporter plasmids, such as cryptic promoter (31) and/or splicing activities, cells were transfected with in vitro capped reporter RNA constructs. RNA transfections were performed using the wild-type and M9 recombinant 18S rRNAs.…”

Section: Resultsmentioning

confidence: 99%

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Matsuda

Mauro

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…It appears that the same general feature that produces, or is suggestive of, IRES activity (GC richness, which promotes RNA secondary structure) also increases the potential for the DNA to act as a cryptic promoter (perhaps by increasing the occurrence of binding sites for transcription factors such as SP1, SP3, AP1, and NFKB, which bind to GC-rich sequences). Even very well authenticated IRESs, such as the IRES from hepatitis C virus, can give rise to considerable cryptic promoter activity (Dumas et al 2003). The cryptic promoters may use heterogeneous start sites, as is sometimes the case with GC-rich promoters that do not contain a TATA box.…”

Section: Discussionmentioning

confidence: 99%

Assessing IRES activity in the HIF-1α and other cellular 5′ UTRs

Bert

Grépin²,

Vadas³

et al. 2006

RNA

113

112

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Dicistronic reporter plasmids, such as the dual luciferase-containing pR-F plasmid, have been widely used to assay cellular and viral 59 untranslated regions (UTRs) for IRES activity. We found that the pR-F dicistronic reporter containing the 59 UTRs from HIF-1a, VEGF, c-myc, XIAP, VEGFR-1, or Egr-1 UTRs all produce the downstream luciferase predominantly as a result of cryptic promoter activity that is activated by the SV40 enhancer elements in the plasmid. RNA transfection experiments using dicistronic or uncapped RNAs, which avoid the complication of cryptic promoter activity, indicate that the HIF-1a, VEGF, c-myc, and XIAP UTRs do have some IRES activity, although the activity was much less than that of the viral EMCV IRES. The translation of transfected monocistronic RNAs containing these cellular UTRs was greatly enhanced by the presence of a 59 cap, raising questions as to the strength or mechanism of IRES-mediated translation in these assays.

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“…To take into account the putative promoter IRES activity from DNA, as reported earlier (Dumas et al, 2003), we used capped RNA transcripts corresponding to different bicistronic DNAs to transfect Huh7 cells and measured the resulting luciferase activities. As shown in Table 2, they were lower than those obtained in vitro for all three genotypes, but there was no significant difference between them.…”

Section: Translation Activity From Genotypes 1 Andmentioning

confidence: 99%

Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

Masante

Mahias

Lourenco

et al. 2008

Journal of General Virology

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Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 59 untranslated region: correlation with reduced in vitro replication Computer analysis of 158 hepatitis C virus (HCV) 59 untranslated region (59 UTR) sequences from the six genotypes showed that the 59 UTR from genotype 3 displays seven specific non-contiguous nucleotide changes, at positions 8, 13, 14, 70, 97, 203 and 224. The purpose of this study was to investigate the impact of these changes on translation and replication activities. Indeed, these modifications could alter both the internal ribosome entry site (IRES) present in the 59 UTR of the plus-strand RNA and the 39 end of the minus strand involved in the initiation of plus-strand RNA synthesis. We found that the genotype 3-specific nucleotide changes do not modify the in vitro or ex vivo translation activity of the corresponding IRES, in comparison with that of genotype 1. In contrast, in vitro replication from the minus-strand RNA is eight times less efficient for genotype 3 than for genotype 1 RNA, suggesting the involvement of some nucleotide changes in the reduction of RNA synthesis. Nucleotides 13, 14 and 224 were found to be responsible for this effect. Moreover, a reduced replicative activity was confirmed ex vivo for genotype 3, but to a lesser extent than that observed in vitro, using an RNA minigenome. INTRODUCTIONHepatitis C virus (HCV) affects nearly 200 million people worldwide. Five to seven per cent of patients die as a consequence of liver disease. Hepatic steatosis is a common feature of liver biopsy specimens from patients with chronic hepatitis C, and its presence is associated with fibrotic progression (Rubbia-Brandt et al., 2000). Numerous factors including gender, age of infection, alcohol consumption, exposure to other hepatotoxins and perturbation of lipid metabolism have been identified as determinants of pathogenesis. Viral factors may also be critical determinants of steatosis in chronic hepatitis C, particularly HCV genotype (Rubbia-Brandt et al., 2004). Genetic variability of the RNA genome has made it possible to distinguish six HCV genotypes and over 70 subtypes (Simmonds et al., 2005). Each of these genotypes displays particular features such as resistance to interferon/ribavirin treatments. Thus, genotype 1-infected patients respond less efficiently to therapy than those infected with genotype 2 and 3 viruses. Conversely, patients with HCV genotype 3 infection and chronic hepatitis C are more likely to be subjected to a liver steatosis than those infected with HCV genotype 1 (Lonardo et al., 2006). Viral determinants of this differential progression are as yet poorly understood. It has been suspected that sequence variations in the envelope E1 and E2 glycoproteins might be involved in differences in pathogenesis between genotypes 1 and 3 (Shaw et al., 2003). More recently, in vitro models suggested involvement of the core protein as a viral factor associated with lipid accumulation in genotype 3 infection (Abid et al., 2005;Hourioux et al., 20...

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A promoter activity is present in the DNA sequence corresponding to the hepatitis C virus 5' UTR

Cited by 47 publications

References 22 publications

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo

Assessing IRES activity in the HIF-1α and other cellular 5′ UTRs

Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

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