Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA

Abstract

The RNA genome of hepatitis C virus (HCV) terminates with a highly conserved 98-base sequence. Enzymatic and chemical approaches were used to define the secondary structure of this 3-terminal element in RNA transcribed in vitro from cloned cDNA. Both approaches yielded data consistent with a stable stem-loop structure within the 3-terminal 46 bases. In contrast, the 5 52 nucleotides of this 98-base element appear to be less ordered and may exist in multiple conformations. Under the experimental conditions tested, interaction between the 3 98 bases and upstream HCV sequences was not detected. These data provide valuable information for future experiments aimed at identifying host and/or viral proteins which interact with this highly conserved RNA element. MATERIALS AND METHODS Construction of plasmids for RNA synthesis. The full-length HCV consensus sequence (genotype 1a) was cloned downstream of the T7 RNA polymerase promoter in a pBR322 derivative, pTET, where the sequences between DraI-EcoRI were deleted and replaced with a polylinker (20) (Fig. 1). The sequence between the ApaI sites in the ORF were deleted, leaving the entire 5Ј and 3Ј NTRs (⌬Apa [Fig. 1]). A deletion between XcmI and MseI in the ⌬Apa plasmid created a construct containing 9 nt of the 5Ј NTR and 4 nt of the poly(U/UC) tract upstream of the 98-base element (⌬Xcm [Fig. 1]). The fourth construct contained only 52 nt of the 98-base element (⌬SL I [Fig. 1]). Plasmid DNAs were linearized with either BsmI or EcoRV such that transcribed RNA would presumably have the same 3Ј terminus as HCV RNA or additional nonviral sequences to serve as a primer binding site during cDNA synthesis, respectively (Fig. 1). RNA was transcribed according to standard procedures, using T7 RNA polymerase (Epicentre). Radioactive end labeling. Dephosphorylated in vitro-transcribed RNA and gel-purified oligonucleotides were 5Ј end labeled by using T4 polynucleotide kinase (Gibco-BRL) and [␥-32 P]ATP (6,000 Ci/mmol; Amersham). Dephosphorylated RNA and oligonucleotides were incubated in a 20-l reaction mixture containing 70 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 100 mM KCl, 1 mM 2-mercaptoethanol, 100 Ci of [␥-32 P]ATP, and 10 U of T4 polynucleotide kinase for 30 min at 37°C. The reaction mixture was phenol-chloroform extracted and ethanol precipitated. Labeling at the 3Ј end of RNA was performed by ligation of [␣-32 P]pCp (3,000 Ci/mmol; Amersham) with T4 RNA ligase (Gibco-BRL) as described by England and Uhlenbeck (10). All radiolabeled RNA was purified by denaturing polyacrylamide gel electrophoresis, eluted in 100 mM Tris-HCl (pH 8.0)-1 mM EDTA, phenol-chloroform extracted, and ethanol precipitated. Structure-specific enzymatic probing. Enzymatic probing of in vitro-transcribed RNAs was performed at 0°C for 30 min with the following RNases: cobra venom RNase V 1 (0.01 and 0.1 U; Pharmacia), RNase CL3 (0.1 U; U.S. Biochemical [USB]), and RNases T 1 (USB), T 2 (Gibco-BRL), Phy M (Gibco-BRL), and B. cereus (USB) (each at 1 U). RNase digestion of 10 5 cpm of 32 P-labeled RNA was ...

5′- and 3′-noncoding regions in flavivirus RNA

“…Similar stem-loop secondary structures with similar predicted thermal stability have been subsequently proposed for the 3 0 termini of all flavivirus genomes for which nucleotide sequence data are available (see, e.g., Brinton et al, 1986;Hahn et al, 1987;Irie et al, 1989;Wengler and Castle, 1986). In fact, similar structures are predicted to form at the 3 0 termini of the genomes of the other Flaviviridae, the hepaci-and pestiviruses (Blight and Rice, 1997;Deng and Brock, 1993). The stem consists of about 30 hydrogen-bonded base pairs and is variably interrupted by bulges due to the predicted apposition of nucleotides that are unable to hydrogen bond.…”

Hepatitis C Virus RNA Replication

“…Two copies of miR-122 binding to the 5′NTR are shown in grey. b 3′end of the viral positive strand (Blight and Rice 1997). Long range interactions of SL3.2 with sequences around 9,110 (Tuplin et al 2012) and with the loop region of SL2 (Friebe et al 2005) are indicated by arrows.…”

“…The 98-nt X-tail is almost invariant among HCV isolates and is supposed to contain the main regulatory elements required for negative-strand synthesis (Kolykhalov et al 1996;Tanaka et al 1995). It comprises three experimentally validated stem-loop structures (Blight and Rice 1997), which are all essential for viral replication (Friebe and Bartenschlager 2002;Yi and Lemon 2003b;Yi and Lemon 2003a) and barely tolerate mutations (Yi and Lemon 2003b;Yi and Lemon 2003a), indicating that not only the structures, but also the sequences are critical for RNA replication. The very 3′end of the HCV genome is a uridine residue in all HCV isolates analyzed so far and base paired in the very stable 3′-terminal SL1 (Fig.…”

RNA chaperones encoded by RNA viruses

“…The family Flaviviridae comprises three genera, Flavivirus, Pestivirus and Hepacivirus (Shepard et al, 2005) The genomic RNA 5′UTR and 3′UTR of HCV contain a number of structured RNA elements that have been reported to be involved in viral replication (Ito and Lai, 1997;Friebe et al, 2005;Song et al, 2006). The HCV 5′-UTR contains 341 nucleotides (nt) and is folded into an internal ribosome entry site (IRES) structure that mediates cap-independent translation, while its 3′-UTR is 230-nt long and contains three structured RNA elements, including a variable sequence with VSL1 and VSL2 stem-loops, a poly U/UC tract, and a 98-nt 3′X region that forms three stem-loops (Kolykhalov et al, 1996;Blight and Rice, 1997;Friebe and Bartenschlager, 2002). On cleavage, the HCV polyprotein yields three structural proteins, named core protein, E1 and E2.…”