3′ RNA Elements in Hepatitis C Virus Replication: Kissing Partners and Long Poly(U)

ABSTRACT The hepatitis C virus (HCV) genomic RNA possesses conserved structural elements that are essential for its replication. The 3′ nontranslated region (NTR) contains several of these elements: a variable region, the poly(U/UC) tract, and a highly conserved 3′ X tail, consisting of stem-loop 1 (SL1), SL2, and SL3. Studies of drug-selected, cell culture-adapted subgenomic replicons have indicated that an RNA element within the NS5B coding region, 5BSL3.2, forms a functional kissing-loop tertiary structure with part of the 3′ NTR, 3′ SL2. Recent advances now allow the efficient propagation of unadapted HCV genomes in the context of a complete infectious life cycle (HCV cell culture [HCVcc]). Using this system, we determine that the kissing-loop interaction between 5BSL3.2 and 3′ SL2 is required for replication in the genotype 2a HCVcc context. Remarkably, the overall integrity of the 5BSL3 cruciform is not an absolute requirement for the kissing-loop interaction, suggesting a model in which trans-acting factor(s) that stabilize this interaction may interact initially with the 3′ X tail rather than 5BSL3. The length and composition of the poly(U/UC) tract were also critical determinants of HCVcc replication, with a length of 33 consecutive U residues required for maximal RNA amplification. Interrupting the U homopolymer with C residues was deleterious, implicating a trans-acting factor with a preference for U over mixed pyrimidine nucleotides. Finally, we show that both the poly(U) and kissing-loop RNA elements can function outside of their normal genome contexts. This suggests that the poly(U/UC) tract does not function simply as an unstructured spacer to position the kissing-loop elements.

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cis-Acting RNA Signals in the NS5B C-Terminal Coding Sequence of the Hepatitis C Virus Genome

Journal of Virology ◽

10.1128/jvi.78.20.10865-10877.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 10865-10877 ◽

Cited By ~ 67

Author(s):

Haekyung Lee ◽

Hyukwoo Shin ◽

Eckard Wimmer ◽

Aniko V. Paul

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Genome ◽

Coding Region ◽

Subgenomic Replicon ◽

Stem Loop ◽

Coding Sequence ◽

Cis Acting ◽

Rna Elements

ABSTRACT The cis-replicating RNA elements in the 5′ and 3′ nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3′ NTR RNA, and only poorly by 5′ NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.

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Role of the Hepatitis C Virus Core+1 Open Reading Frame and Core cis-Acting RNA Elements in Viral RNA Translation and Replication

Journal of Virology ◽

10.1128/jvi.01640-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11503-11515 ◽

Cited By ~ 68

Author(s):

Niki Vassilaki ◽

Peter Friebe ◽

Philipe Meuleman ◽

Stephanie Kallis ◽

Artur Kaul ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Core Gene ◽

Stem Loop ◽

Reading Frame ◽

Rna Translation ◽

The Core ◽

Rna Elements

ABSTRACT Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.

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Inhibition of hepatitis C virus replication by pol III-directed overexpression of RNA decoys corresponding to stem-loop structures in the NS5B coding region

Virology ◽

10.1016/j.virol.2005.08.003 ◽

2005 ◽

Vol 342 (2) ◽

pp. 276-285 ◽

Cited By ~ 19

Author(s):

Jing Zhang ◽

Osamu Yamada ◽

Takashi Sakamoto ◽

Hiroshi Yoshida ◽

Hiromasa Araki ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Replication ◽

Coding Region ◽

Stem Loop ◽

Pol Iii

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Genetic Analysis of Sequences in the 3′ Nontranslated Region of Hepatitis C Virus That Are Important for RNA Replication

Journal of Virology ◽

10.1128/jvi.76.11.5326-5338.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5326-5338 ◽

Cited By ~ 196

Author(s):

Peter Friebe ◽

Ralf Bartenschlager

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatoma Cell ◽

Rna Stability ◽

Variable Region ◽

Rna Replication ◽

Hepatoma Cell Line ◽

Human Hepatoma Cell ◽

Stem Loop ◽

Nontranslated Region

ABSTRACT The genome of the hepatitis C virus (HCV) is a plus-strand RNA molecule that carries a single long open reading frame. It is flanked at either end by highly conserved nontranslated regions (NTRs) that mediate crucial steps in the viral life cycle. The 3′ NTR of HCV has a tripartite structure composed of an about 40-nucleotide variable region, a poly(U/UC) tract that has a heterogeneous length, and a highly conserved 98-nucleotide 3′-terminal sequence designated the X tail or 3′X. Conflicting data as to the role the sequences in the 3′ NTR play in RNA replication have been reported. By using the HCV replicon system, which is based on the self-replication of subgenomic HCV RNAs in human hepatoma cell line Huh-7, we mapped in this study the sequences in the 3′ NTR required for RNA replication. We found that a mutant with a complete deletion of the variable region is viable but that replication is reduced significantly. Only replicons in which the poly(U/UC) tract was replaced by a homouridine stretch of at least 26 nucleotides were able to replicate, whereas RNAs with homopolymeric guanine, adenine, or cytosine sequences were inactive. Deletions of individual or all stem-loop structures in 3′X were not tolerated, demonstrating that this region is most crucial for efficient RNA replication. Finally, we found that none of these deletions or substitutions within the 3′ NTR affected RNA stability or translation, demonstrating that the primary effect of the mutations was on RNA replication. These data represent the first detailed mapping of sequences in the 3′ NTR assumed to act as a promoter for initiation of minus-strand RNA synthesis.

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RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand

Journal of General Virology ◽

10.1099/vir.0.016907-0 ◽

2010 ◽

Vol 91 (5) ◽

pp. 1207-1212 ◽

Cited By ~ 6

Author(s):

H. Kanamori ◽

K. Yuhashi ◽

S. Ohnishi ◽

K. Koike ◽

T. Kodama

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Loop Structure ◽

Coding Region ◽

Rna Dependent Rna Polymerase ◽

Stem Loop ◽

Negative Strand ◽

Stem Loop Structure

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Role of RNA Structures in Genome Terminal Sequences of the Hepatitis C Virus for Replication and Assembly

Journal of Virology ◽

10.1128/jvi.01508-09 ◽

2009 ◽

Vol 83 (22) ◽

pp. 11989-11995 ◽

Cited By ~ 41

Author(s):

Peter Friebe ◽

Ralf Bartenschlager

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Virus ◽

Hcv Rna ◽

Rna Structures ◽

Rna Packaging ◽

Stem Loop ◽

Nontranslated Region ◽

Positive Strand Rna Virus ◽

Positive Strand Rna

ABSTRACT Hepatitis C virus (HCV) is a positive-strand RNA virus replicating its genome via a negative-strand [(−)] intermediate. Little is known about replication signals residing in the 3′ end of HCV (−) RNA. Recent studies identified seven stem-loop structures (SL-I′, -IIz′, -IIy′, -IIIa′, -IIIb′, -IIIcdef′, and -IV′) in this region. In the present study, we mapped the minimal region required for RNA replication to SL-I′ and -IIz′, functionally confirmed the SL-IIz′ structure, and identified SL-IIIa′ to -IV′ as auxiliary replication elements. In addition, we show that the 5′ nontranslated region of the genome most likely does not contain cis-acting RNA structures required for RNA packaging into infectious virions.

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