Secondary structure of the 5′ nontranslated regions of hepatitis C virus and pestivirus genomic RNAs

Abstract

The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5' nontranslated regions (5'NTRs) sharing short segments of conserved primary nucleotide sequence. The functions of these 5'NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5'NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5'NTRs are highly conserved structurally, despite substantial differences in their primary nucleotide sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5'NTR of HCV resulted in a model of the secondary structure of the HCV 5'NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3' 200 bases of the 5'NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.

Structural analysis of hepatitis C RNA genome using DNA microarrays

“…These regions contains two distinct RNA elements: a short 5 0 proximal RNA element (nt 1-43) that regulates HCV RNA replication and a longer IRES element (nt 44-370) where protein synthesis starts, eventually leading to production of the polyprotein precursor. The results reported here, both with the 361 nt long and the 417 nt long HCV transcripts (Figure 6), fit quite well with the model proposed for a compact IRES element and an accessible region corresponding to the 5 0 proximal RNA element, mainly to nucleotides 24-44 (35,37,53,54).…”

“…Biochemical and functional studies have established that the HCV 5 0 NCR folds into a highly ordered complex structure with multiple stem-loops (35,37,53,54). These regions contains two distinct RNA elements: a short 5 0 proximal RNA element (nt 1-43) that regulates HCV RNA replication and a longer IRES element (nt 44-370) where protein synthesis starts, eventually leading to production of the polyprotein precursor.…”

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus

“…Schematic representation of the secondary structure of the CSFV IRES (based on Brown et al+, 1992;Wang et al+, 1995;Sizova et al+, 1998) showing sites that are either protected from cleavage by RNAses T 1 and V 1 or at which cleavage is enhanced following binding of a 40S ribosomal subunit+ These sites and the position of toeprints caused by bound 40S subunits are indicated by symbols tabulated at the upper right+ The initiation codon ) is underlined+ IRES subdomains are named using nomenclature proposed by Honda et al+ (1996) for the HCV IRES+ The helices that constitute the pseudoknot are labeled 1a, 1b, and 2+ 1A, 4A, 4B, and 4F (Pestova et al+, 1998a(Pestova et al+, , 1998b)+ This binding step is very precise so that the initiation codon is positioned in the immediate vicinity of the ribosomal P site (Pestova et al+, 1998b) and results from specific interactions between elements of the IRES and components of the 43S complex+ Two such interactions have been identified in addition to codon-anticodon base pairing+ The apical half of domain III is bound by eIF3 (Buratti et al+, 1998;Pestova et al+, 1998b;Sizova et al+, 1998;Odreman-Macchioli et al+, 2000)+ In addition, these IRESs contain as yet undefined determinants that mediate factor-independent binding of 40S subunits at the initiation codon (Pestova et al+, 1998b;)+ The ability to bind directly to the small ribosomal subunit is highly unusual among eukaryotic mRNAs and has, to date, been reported only for CSFV-like IRESs and, very recently, for the unrelated IRES in the intergenic region of Cricket paralysis virus (Wilson et al+, 2000)+ Direct binding of small (30S) ribosomal subunits to mRNA through ShineDalgarno base pairing is characteristic of initiation in prokaryotes (Shine & Dalgarno, 1974)+ However, binding of eukaryotic 40S subunits to CSFV-like IRESs is stabilized by multiple IRES-40S subunit interactions and is thus not directly analogous to the prokaryotic interaction+ For example, primer extension on the CSFV IRES is arrested by a bound 40S subunit at C 334 in the pseudoknot and at UUU 387-389 downstream of the initiation codon+ This observation suggests that the initiation codon and flanking residues are fixed in the mRNAbinding cleft of the 40S subunit, and that the 40S subunit binds the IRES at one or more additional positions+…”

Structure of the full-length HCV IRES in solution

“…S1) 13 . The molecule contains tertiary fold motifs, the IIIabc four-way junction and the IIIef/IV pseudoknot 4,14,15 , and adopts a defined ion-dependent fold under physiological conditions 16 , although it does not form a compact, globular structure 13,16,17 . The structure at atomic resolution has been determined for several autonomous folding regions, including domain II, the III abc junction and, more recently, the III ef /IV pseudoknot [18][19][20][21][22][23][24] .…”