Mutational analysis of a conserved tetraloop in the 5′ untranslated region of hepatitis C virus identifies a novel RNA element essential for the internal ribosome entry site function

Psaridi, L; Georgopoulou, Urania; Varaklioti, Agoritsa; Mavromara, Penelope

doi:10.1016/s0014-5793(99)00662-6

Cited by 48 publications

(51 citation statements)

References 39 publications

(71 reference statements)

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“…Studies on domain IIIe of the HCV IRES element have indicated that each of the nucleotides within the highly conserved GAUA tetraloop is crucial for HCV IRES activity (22,31). This domain, together with domain IIId, plays an essential role in binding the 40S ribosomal subunit (18,25,39).…”

Section: Resultsmentioning

confidence: 99%

The Picornavirus Avian Encephalomyelitis Virus Possesses a Hepatitis C Virus-Like Internal Ribosome Entry Site Element

Bakhshesh

Groppelli

Willcocks

et al. 2008

J Virol

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Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotidelong 5 untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3 end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.Translation initiation on the majority of cellular mRNAs is mediated by a cap-dependent mechanism. The cap structure (m 7 GpppN) found on all cytoplasmic mRNAs is recognized by the translation initiation factor complex eIF4F (reviewed in reference 28). This complex contains three proteins: eIF4E, which is the cap-binding protein; eIF4A, which has RNA helicase activity; and eIF4G, which acts as a protein scaffold between the mRNA and the 40S ribosomal subunit via its interaction with eIF3 (reviewed in reference 14). In contrast, initiation of protein synthesis on some viral mRNAs, for example, from the picornaviruses, occurs by a cap-independent mechanism termed internal initiation. In this case, translation initiation is directed by an internal ribosome entry site (IRES) element located within the 5Ј untranslated region (5ЈUTR) of the viral genome (reviewed in references 2 and 11). These IRES elements are large, typically 450 nucleotides (nt) in length, and contain extensive secondary structure; they have been shown to interact with a variety of cellular proteins (2). Most of these elements work without any requirement for eIF4E and hence can continue to function when cap-dependent protein synthesis is inhibited.The picornavirus IRES elements are divided into several groups which display distinct secondary structures and biological properties. One group (class I) contains IRES elements from the entero-and rhinoviruses (e.g., poliovirus [PV]), while the second group contains the cardio-and aphthovirus IRES elements (e.g., encephalomyocarditis virus [EMCV]). The cardio-/aphthovirus IRES elements function efficiently in the rabbit reticulocyte lysate (RRL) translation system. However, the PV and rhinovirus IRES ele...

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Section: Resultsmentioning

confidence: 99%

The Picornavirus Avian Encephalomyelitis Virus Possesses a Hepatitis C Virus-Like Internal Ribosome Entry Site Element

Bakhshesh

Groppelli

Willcocks

et al. 2008

J Virol

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“…In patient A, two brain-derived sequences (B4 and B23) showed marked reductions in IRES function compared to the consensus sequence (translational activity for B4 ϭ 5%, and translational activity for B23 ϭ 53% [P Ͻ 0.001]). B4 contained two mutations (C to U at position 122 and A to G at position 298), both of which have been shown by mutagenesis to lie in sequence motifs (pyrII and the domain IIIe tetraloop, respectively), which are crucial to IRES function (46,57). Similarly, the single mutation in B23 (U to C at position 259) falls within the domain IIId loop E motif, a highly conserved region, in which mutations are deleterious to IRES function (20).…”

Section: Resultsmentioning

confidence: 99%

“…In patient A, it is interesting that the mutations segregate along the IRES, with the brain sequence mutations all found in domains III and IV, specifically within the pyrII motif, the domain IIIe tetraloop, and the domain IIId loop E motif, which are crucial to IRES function (20,46,57), in addition to the A204, A243 mutation (Fig. 7).…”

Section: Discussionmentioning

confidence: 99%

Identification of Unique Hepatitis C Virus Quasispecies in the Central Nervous System and Comparative Analysis of Internal Translational Efficiency of Brain, Liver, and Serum Variants

Forton

Karayiannis

Mahmud

et al. 2004

J Virol

226

171

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Reports of cerebral dysfunction in chronic hepatitis C virus (HCV) infection have led to the suggestion that HCV may infect the central nervous system (CNS). We used reverse transcription-PCR, cloning, and sequencing to define quasispecies for the HCV internal ribosomal entry site (IRES) and hypervariable region 1 (HVR1) in autopsy-derived brain, liver, lymph node, and serum samples. There was evidence of tissue compartmentalization of sequences in the brain in two patients, with between 24 and 55% of brain-derived IRES sequences absent from the serum, and significant phylogenetic and phenetic clustering of the brain and lymph node HVR1 sequences. The IRES initiates cap-independent translation of the viral polyprotein. Two unique brain-derived IRES mutations (C 204 3A and G 243 3A), which have previously been associated with lymphoid replication and altered translational efficiency in cell culture, were found in one patient. We used a dicistronic reporter vector to test whether brain-derived variants showed altered IRES-mediated translational efficiency, which might favor CNS infection. The translational efficiencies of the brain-derived IRES sequences were generally reduced compared to those of the master serum and liver sequences in rabbit reticulocyte cell lysates and two human cell lines, HuH7 (liver) and CHME3 (microglial). The C 204 3A and G 243 3A mutations showed preserved translational efficiency in HuH7 cells but reduced efficiency in CHME3 cells. Our data provide evidence that the CNS is a site of HCV replication, consistent with the recent demonstration of negative-strand HCV RNA in brain, and suggest that IRES polymorphisms may be important as a viral strategy of reduced translation to favor latency in the CNS.

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“…This IRES shares many characteristics with the HCV IRES, including the presence of a pseudoknot (domain IIIf) and a highly conserved stem-loop (domain IIIe), which plays a critical role in the interaction with the 40S ribosomal subunit (Psaridi et al 1999;Lukavsky et al 2000;Kieft et al 2001;Laletina et al 2006). We have now constructed a series of plasmids containing some of these variants of the PTV-1 IRES; the cDNA (z300 nt) was inserted as a BamHI fragment into the pRBRluc vector (Fig.…”

Section: Defective Ptv Ires Elements Do Not Inhibit Translation Of Momentioning

confidence: 99%

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism

Belsham¹,

Nielsen²,

Normann³

et al. 2008

RNA

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The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 59 cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 59 untranslated region (59UTR), contain extensive secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which are unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 59UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces cleavage of eIF4G) and is also inhibited by hippuristanol, a specific inhibitor of eIF4A function, in contrast to their parental wild-type IRES elements. These results provide a possible basis for the evolution of viral IRES elements within the context of functional mRNAs that are translated by a cap-dependent mechanism.

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Mutational analysis of a conserved tetraloop in the 5′ untranslated region of hepatitis C virus identifies a novel RNA element essential for the internal ribosome entry site function

Cited by 48 publications

References 39 publications

The Picornavirus Avian Encephalomyelitis Virus Possesses a Hepatitis C Virus-Like Internal Ribosome Entry Site Element

The Picornavirus Avian Encephalomyelitis Virus Possesses a Hepatitis C Virus-Like Internal Ribosome Entry Site Element

Identification of Unique Hepatitis C Virus Quasispecies in the Central Nervous System and Comparative Analysis of Internal Translational Efficiency of Brain, Liver, and Serum Variants

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5′ untranslated regions are efficiently translated in cells by a cap-dependent mechanism

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