Internal Ribosome Entry Site-Mediated Translation in Hepatitis C Virus Replication

Rijnbrand, René; Lemon, Stanley M.

doi:10.1007/978-3-642-59605-6_5

Cited by 77 publications

(90 citation statements)

References 116 publications

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“…Although the proposed structure was generated by using only the HCV 1a genome sequence, without the aid of phylogenetic comparison, it appears to be applicable to other HCV strains and genotypes. The negative-strand folding is compatible with observed patterns of nucleotide covariance that were used previously to define the positive-strand 5Ј-UTR structure (40). Four consecutive covariant base pairs (178 to 181:218 to 221) within subdomain IIIb are retained.…”

supporting

confidence: 68%

“…(B) Alternative base-pairing configuration for nt 1 to 20. (C) Positive-strand 5Ј-UTR/core region (nt 1 to 365) secondary-structure domains (40). An RNA pseudoknot structure, psk, is formed by base-pairing of subdomain IIIf and sequences upstream of domain IV.…”

Section: Vol 76 2002 Nuclease Mapping Of Hcv 3ј Termini 9565mentioning

confidence: 99%

“…The 341-nucleotide (nt) 5Ј-UTR of the positive strand acts as an internal ribosome entry site (IRES) to direct cap-independent translation of the single ϳ3,000-codon-long HCV open reading frame (38,40). The tripartite 3Ј-UTR consists of a short upstream variable region, a central poly(U)-polypyrimidine stretch of variable length, and a terminal highly conserved 98-nt sequence (38).…”

mentioning

confidence: 99%

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Secondary structure and hybridization accessibility of the hepatitis C virus negative strand RNA 5′‐terminus

Smith

2004

Journal of Viral Hepatitis

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The 3 -terminal sequences of hepatitis C virus (HCV) positive-and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3 untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3 terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3 -terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positivestrand 3 -X region RNA in vitro.The untranslated regions (UTRs) of the hepatitis C virus (HCV) positive-strand genome and negative-strand intermediate contain cis-acting sequences essential for viral translation and RNA replication. The 341-nucleotide (nt) 5Ј-UTR of the positive strand acts as an internal ribosome entry site (IRES) to direct cap-independent translation of the single ϳ3,000-codon-long HCV open reading frame (38, 40). The tripartite 3Ј-UTR consists of a short upstream variable region, a central poly(U)-polypyrimidine stretch of variable length, and a terminal highly conserved 98-nt sequence (38). With the exception of the variable region, all sequence elements in the 3Ј-UTR are necessary for intracellular replication of HCV RNA (16,29,56).A number of in vitro studies (26,35,36,39,58) have established that the terminal 98-nt X region sequence can serve as a minimal template for de novo initiation of negative-strand synthesis by the viral RNA-dependent RNA polymerase, NS5B. The 3Ј terminus of the HCV negative strand reportedly plays an analogous role in positive-strand RNA synthesis (25,36,39). The binding of HCV 3Ј termini to various host proteins may exert subtle effects on IRES-mediated translation (16,23,34,55) or protect viral transcripts from degradation by cytoplasmic RNases (15,48,49).The replicative and protein-binding functions of heteropolymeric regions in the HCV 3Ј termini are, in many instances, dependent on the ability of the primary sequence to fold into higher-order RNA structure. In vitro, the X region sequence is capable of adopting a three-stem-loop structure, with the 3Ј-terminal 46 nt forming a thermodynamically stable stem-loop, SL I (6). Mutational analysis indicates that the duplex structure forming the base of SL I influences both the site and efficiency of de novo initiation by NS5B (26,35). Preservation of the interior stem-...

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supporting

confidence: 68%

Section: Vol 76 2002 Nuclease Mapping Of Hcv 3ј Termini 9565mentioning

confidence: 99%

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confidence: 99%

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Secondary structure and hybridization accessibility of the hepatitis C virus negative strand RNA 5′‐terminus

Smith

2004

Journal of Viral Hepatitis

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“…Eukaryotic cells and their viruses have evolved at least two mechanisms for recruiting and positioning ribosomes at the start sites for translation of RNA messages+ The primary mechanism involves recognition of a 7-methyl guanosine cap on the 59 terminus of the mRNA by a set of canonical initiation factors that recruit the 43S particle-including the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3)-forming the 48S preinitiation complex (Fig+ 1A; for review, see Merrick & Hershey, 1996;Pain, 1996;Sachs et al+, 1997)+ Alternatively, numerous viruses and some eukaryotic mRNAs utilize a cap-independent pathway in which an RNA element, the internal ribosome entry site (IRES), drives preinitiation complex formation by positioning the ribosome on the message, either at or just upstream of the start site+ In hepatitis C virus (HCV), the major infectious agent leading to non-A, non-B hepatitis, the minimum IRES includes nearly the entire 59 untranslated region (UTR) of the message (for review, see Rijnbrand & Lemon, 2000)+ The secondary structure of the HCV IRES RNA, one of the most conserved regions of the entire viral genome, is critical for translation initiation, and is similar to that of the related pestiviruses and GB virus B (Brown et al+, 1992;Wang et al+, 1994Wang et al+, , 1995Le et al+, 1995;Rijnbrand et al+, 1995;Honda et al+, 1996aHonda et al+, , 1996bHonda et al+, , 1999Pickering et al+, 1997;Varaklioti et al+, 1998;Psaridi et al+, 1999;Tang et al+, 1999) + We have previously shown that the HCV IRES RNA adopts a specific three-dimensional fold in the presence of physiological concentrations of metal ions (Kieft et al+, 1999)+ Rather than forming a tightly packed globular structure, the RNA helices extend from two folded helical junctions, JIIIabc and JIIIef (Fig+ 1B)+ This suggests that the IRES RNA acts as a structural scaffold in which specifically placed recognition sites recruit the translational machinery+ This is supported by the observation that eIF3 and the 40S ribosomal subunit, the two largest components of the 43S particle, bind directly to the HCV IRES RNA (Pestova et al+, 1998)+ Unlike IRESs found in some other RNA viruses, such as poliovirus, the IRES RNA•40S•eIF3 ternary pre-initiation complex forms without the involvement of other cellular factors (Fig+ 1A;Pestova et al+, 1998)+ Although several other proteins appear to interact with the HCV IRES RNA, they are not required for 43S binding to the IRES …”

Section: Introductionmentioning

confidence: 99%

Mechanism of ribosome recruitment by hepatitis C IRES RNA

Kieft

Zhou

Jubin³

et al. 2001

RNA

341

487

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Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 59 untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA•40S•eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

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“…This indicates the impact of host trans-acting factors in the modulation of IRES activity. Such host factors as polypyrimidine tracting-binding protein (PTB), La autoantigen, some ribonucleoproteins, and microRNA122 have been identified as modulators of IRES activity (24)(25)(26).…”

Section: Original Articlementioning

confidence: 99%

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

et al. 2011

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The initiation of translation in hepatitis C virus (HCV) occurs at the internal ribosome entry site (IRES) located at the 5 0 -end of its genomic RNA. To study the function of HCV IRES, we constructed a reporter plasmid that generates a bicistronic mRNA encoding two fluorescent proteins: cap-dependent DsRed2 and IRES-dependent Azami Green (AG). We introduced the plasmid into Huh7.5.1 and HEK293 cells and measured the relative IRES activity from the ratio of AG's signal to DsRed2's in individual cells using flow cytometry. To compare our method and a conventional biochemical method, we constructed a structurally similar reporter in which Renilla and Firefly luciferases replace DsRed2 and AG, respectively. With these systems, we found that the IRES A164G substitution decreased its activity, that interferon alpha affected the IRES activity in a cell type-specific manner, and that a synthetic micro-RNA targeting IRES was able to suppress the gene expression. In conclusion, the two methods were comparable in sensitivity in the studies of IRES mutations and host cell types. We discussed the significance of our findings and potential advantage of the cytometric assay: application to the molecular study of the HCV translation and to screening anti-IRES drugs. ' 2011 International Society for Advancement of Cytometry Key terms hepatitis C virus; internal ribosome entry site; DsRed2; Azami Green; flow cytometry HEPATITIS C virus (HCV) is a major causative agent of liver cirrhosis and hepatocellular carcinoma (1). The HCV genome is a plus-strand RNA consisting of $9,600 nucleotides (nt) and has a single long open reading frame encoding a polyprotein of about 3,000 amino acids (2). Unlike the translation of eukaryotic mRNA starting from the 5 0 -cap structure, the translation of HCV RNA initiates at the internal ribosome entry site (IRES), which encompasses most of the 5 0 -untranslated region (5 0 -UTR) and about 40 nt of the core protein-coding region (3,4). The HCV IRES is highly conserved among HCV isolates in both the primary sequence and secondary structure (5-7). Since the translation is an essential step in the viral replication and possibly in the pathogenic functions, it would be important to evaluate the activity of HCV IRES for such studies.The HCV IRES activity has been studied, by using biochemical methods with luciferase genes as reporters (8-10), which have some flaws resulting from using cell lysates. In this study we applied flow cytometry to the IRES studies, in an attempt to simplify the assay procedure and to expand the scope of the study. Thus, we used a combination of two fluorescent proteins, DsRed2 (11) and Azami-Green (AG) (12), which are excited by the light of the identical wave length and whose emission lights are easily discriminated. We constructed and used a bicistronic reporter plasmid with genes encoding these two fluorescent proteins, and by flow cytometry, quantitatively determined fluorescent signals in individual cells transfected with the plasmid. To

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Internal Ribosome Entry Site-Mediated Translation in Hepatitis C Virus Replication

Cited by 77 publications

References 116 publications

Secondary structure and hybridization accessibility of the hepatitis C virus negative strand RNA 5′‐terminus

Secondary structure and hybridization accessibility of the hepatitis C virus negative strand RNA 5′‐terminus

Mechanism of ribosome recruitment by hepatitis C IRES RNA

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

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