Genetic Analysis of a Poliovirus/Hepatitis C Virus Chimera: New Structure for Domain II of the Internal Ribosomal Entry Site of Hepatitis C Virus

Lee

et al. 2003

RNA

Hepatitis C virus (HCV) is a positive-sense RNA virus ∼9600 bases long. An internal ribosomal entry site (IRES) spans the 5 nontranslated region, which is the most conserved and highly structured region of the HCV genome. In this study, we demonstrate that nucleotides 428-442 of the HCV core-coding sequence anneal to nucleotides 24-38 of the 5NTR, and that this RNA-RNA interaction modulates IRES-dependent translation in rabbit reticulocyte lysate and in HepG2 cells. The inclusion of the core-coding sequence (nucleotides 428-442) significantly suppressed the translational efficiency directed by HCV IRES in dicistronic reporter systems, and this suppression was relieved by site-directed mutations that blocked the long-range interaction between nucleotides 24-38 and 428-442. These findings suggest that the long-range interaction between the HCV 5NTR and the core-coding nucleotide sequence down-regulate cap-independent translation via HCV IRES. The modulation of protein synthesis by long-range RNA-RNA interaction may play a role in the regulation of viral gene expression.

Section: Discussionmentioning

confidence: 99%

Long-range RNA–RNA interaction between the 5′ nontranslated region and the core-coding sequences of hepatitis C virus modulates the IRES-dependent translation

Lee

et al. 2003

RNA

“…The initial secondary structure had been predicted using both enzymatic probing results as constraints in an early version of the MFOLD program (Zuker and Stiegler 1981) along with comparative computer-predicted models with the UTRs of BVDV and CSFV (Brown et al 1992). There have been numerous studies adding to the refinement of the IRES structure (Lemon and Honda 1997;Lyons et al 2001;Odreman-Macchioli et al 2001), arriving at two similar models (Honda et al 1999;Zhao and Wimmer 2001;Lytle et al 2002) with some differences in domain II. Smaller stem-loop motifs of the HCV IRES structure have had their tertiary structure determined using both X-ray crystallography (Kieft et al 2002) and NMR (Klinck et al 2000;Lukavsky et al 2000;Collier et al 2002).…”

Section: Viral Ires Structuresmentioning

confidence: 99%

“…These wet-laboratory determinations of structure can also be supported with phylogenetic data comparisons in which sequences that have changed have preserved the structure to preserve the function of the structure. The best data from all these approaches are used to support a proposed model, but conflicting secondary structure models do arise in the literature as has been evident in domain II of HCV (Honda et al 1999;Zhao and Wimmer 2001;Lytle et al 2002).…”

Section: Ires Structurementioning

confidence: 99%

Searching for IRES

Baird¹,

Turcotte²,

Korneluk³

et al. 2006

RNA

276

270

The cell has many ways to regulate the production of proteins. One mechanism is through the changes to the machinery of translation initiation. These alterations favor the translation of one subset of mRNAs over another. It was first shown that internal ribosome entry sites (IRESes) within viral RNA genomes allowed the production of viral proteins more efficiently than most of the host proteins. The RNA secondary structure of viral IRESes has sometimes been conserved between viral species even though the primary sequences differ. These structures are important for IRES function, but no similar structure conservation has yet to be shown in cellular IRES. With the advances in mathematical modeling and computational approaches to complex biological problems, is there a way to predict an IRES in a data set of unknown sequences? This review examines what is known about cellular IRES structures, as well as the data sets and tools available to examine this question. We find that the lengths, number of upstream AUGs, and %GC content of 59-UTRs of the human transcriptome have a similar distribution to those of published IRES-containing UTRs. Although the UTRs containing IRESes are on the average longer, almost half of all 59-UTRs are long enough to contain an IRES. Examination of the available RNA structure prediction software and RNA motif searching programs indicates that while these programs are useful tools to fine tune the empirically determined RNA secondary structure, the accuracy of de novo secondary structure prediction of large RNA molecules and subsequent identification of new IRES elements by computational approaches, is still not possible.

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

“…In hepatitis C virus (HCV), a human pathogen and world-wide health threat, the minimal sequence and secondary structure requirements of IRES-driven translation have been defined (6)(7)(8)(9)(10), the presence and architecture of an IRES RNA tertiary fold has been described (11,12), and the identities and binding sites of necessary cofactors have been determined (12,13). Because the IRES RNA functionally replaces the eukaryotic initiation factor (eIF) 4F protein complex (eIF4E, eIF4G, and eIF4A), translation initiation in HCV requires just two of the canonical initiation factors, eIF2 and eIF3, for correct positioning of the initiator tRNA during cap-independent initiation (13).…”

mentioning

confidence: 99%

Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA

Fraser

et al. 2004

159

206

Protein synthesis in all cells begins with recruitment of the small ribosomal subunit to the initiation codon in a messenger RNA. In some eukaryotic viruses, RNA upstream of the coding region forms an internal ribosome entry site (IRES) that directly binds to the 40S ribosomal subunit and enables translation initiation in the absence of many canonical translation initiation factors. The hepatitis C virus (HCV) IRES RNA requires just two initiation factors, eukaryotic initiation factor (eIF) 2 and eIF3, to form preinitiation 48S ribosomal complexes that subsequently assemble into translation-competent ribosomes. Using an RNA-based affinity purification approach, we show here that HCV IRES RNA facilitates eIF2 function through its interactions with eIF3 and the 40S ribosomal subunit. Although the wild-type IRES assembles normally into 48S and 80S ribosomal complexes in human cell extract, mutant IRES RNAs become trapped at the 48S assembly stage. Trapped 48S complexes formed by IRES mutants with reduced eIF3 binding affinity nonetheless contain eIF3, consistent with inherent eIF3-40S subunit affinity. Intriguingly, however, one of these IRES mutants prevents stable association of both eIF3 and eIF2, preventing initiator tRNA deposition and explaining the block in 80S assembly. In contrast, an IRES mutant unable to induce a conformational change in the 40S subunit, as observed previously by single-particle cryoelectron microscopy, blocks 80S formation at a later stage in assembly. These data suggest that the IRES RNA coordinates interactions of eIF3 and eIF2 on the ribosome required to position the initiator tRNA on the mRNA in the ribosomal peptidyl-tRNA site (P site).eukaryotic initiation factor I nitiation of protein synthesis in eukaryotes requires the ordered assembly of ribosomal preinitiation complexes, beginning with the association of the small (40S) ribosomal subunit with an mRNA (reviewed in refs. 1-3). Cap-dependent translation involves initiation factor protein association with the 7-methyl guanosine moiety at the mRNA 5Ј end, leading to 40S ribosome binding and scanning to the initiation codon before association with the 60S ribosomal subunit to form an active 80S ribosome (1). An alternate pathway, called internal translation initiation, is a cap-independent mechanism of recruiting, positioning, and activating the eukaryotic protein synthesis machinery, driven by structured RNA sequences called internal ribosome entry sites (IRESs) located in the mRNA 5Ј untranslated region (UTR). These sequences have been identified in numerous viral RNAs, and there is evidence suggesting that certain cellular mRNAs may also contain IRES elements (4, 5).In hepatitis C virus (HCV), a human pathogen and world-wide health threat, the minimal sequence and secondary structure requirements of IRES-driven translation have been defined (6-10), the presence and architecture of an IRES RNA tertiary fold has been described (11,12), and the identities and binding sites of necessary cofactors have been determined (12, 13). Becau...