scholarly journals A Hepatitis C Virus cis-Acting Replication Element Forms a Long-Range RNA-RNA Interaction with Upstream RNA Sequences in NS5B

2008 ◽  
Vol 82 (18) ◽  
pp. 9008-9022 ◽  
Author(s):  
Sinéad Diviney ◽  
Andrew Tuplin ◽  
Madeleine Struthers ◽  
Victoria Armstrong ◽  
Richard M. Elliott ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Long Range ◽  
Upstream Region ◽  
Fundamental Aspect ◽  
Genome Replication ◽  
Range Interaction ◽  
Stem Loop ◽  
Long Range Interactions ◽  
Rna Interaction

ABSTRACT The genome of hepatitis C virus (HCV) contains cis-acting replication elements (CREs) comprised of RNA stem-loop structures located in both the 5′ and 3′ noncoding regions (5′ and 3′ NCRs) and in the NS5B coding sequence. Through the application of several algorithmically independent bioinformatic methods to detect phylogenetically conserved, thermodynamically favored RNA secondary structures, we demonstrate a long-range interaction between sequences in the previously described CRE (5BSL3.2, now SL9266) with a previously predicted unpaired sequence located 3′ to SL9033, approximately 200 nucleotides upstream. Extensive reverse genetic analysis both supports this prediction and demonstrates a functional requirement in genome replication. By mutagenesis of the Con-1 replicon, we show that disruption of this alternative pairing inhibited replication, a phenotype that could be restored to wild-type levels through the introduction of compensating mutations in the upstream region. Substitution of the CRE with the analogous region of different genotypes of HCV produced replicons with phenotypes consistent with the hypothesis that both local and long-range interactions are critical for a fundamental aspect of genome replication. This report further extends the known interactions of the SL9266 CRE, which has also been shown to form a “kissing loop” interaction with the 3′ NCR (P. Friebe, J. Boudet, J. P. Simorre, and R. Bartenschlager, J. Virol. 79:380-392, 2005), and suggests that cooperative long-range binding with both 5′ and 3′ sequences stabilizes the CRE at the core of a complex pseudoknot. Alternatively, if the long-range interactions were mutually exclusive, the SL9266 CRE may function as a molecular switch controlling a critical aspect of HCV genome replication.

scholarly journals The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle

2020 ◽  
Vol 21 (4) ◽  
pp. 1479 ◽  
Author(s):  
Cristina Romero-López ◽  
Alfredo Berzal-Herranz
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Long Range ◽  
Genomic Sequence ◽  
Rna Virus ◽  
Hcv Rna ◽  
Structural Elements ◽  
Control Translation ◽  
Rna Interaction

RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.

Long-range RNA–RNA interactions between distant regions of the hepatitis C virus internal ribosome entry site element

2002 ◽  
Vol 83 (5) ◽  
pp. 1113-1121 ◽  
Author(s):  
Esther Lafuente ◽  
Ricardo Ramos ◽  
Encarnación Martínez-Salas
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Long Range ◽  
Internal Ribosome Entry Site ◽  
Translation Efficiency ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Hcv Ires ◽  
Rna Complexes

Efficient internal initiation of translation from the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires sequences of domain II, but the precise role of these sequences is still unknown. In this study, the formation of RNA–RNA complexes in the HCV IRES was evaluated. Using transcripts that contain the sequences of the structural HCV IRES domains II, IIIabcd, IIIabc, IV and IIIef-IV, specific long-range interactions between domains II and IV, as well as domains II and IIIabcd, have been found. These interactions were readily detected in a gel mobility-shift assay and required the presence of magnesium ions. A high concentration of nonspecific competitors, an 80 nt fragment of 18S rRNA or poly(I:C), did not interfere with the formation of RNA complexes. Interestingly, an RNA oligonucleotide bearing the sequence of stem–loop IIId interacted with domain II but not with domain IV or IIIef-IV, strongly suggesting that the interaction between domains II and IIIabcd was mediated by the IIId hairpin. Interaction between domains IIIabcd and IV was barely detected, consistent with the result that the apical part of domain III folds independently of the rest of the IRES. Moreover, the addition of stem–loop IIIef sequences to domain IV significantly reduced its ability to interact, which is in agreement with the formation of a compact RNA structure of domain IV with IIIef. The interactions observed in the absence of proteins between domains II and IV as well as stem–loop IIId and domain II may be transient, having a regulatory role in the translation efficiency of the HCV IRES.

scholarly journals Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3′-Terminal Sequences of Viral Positive- and Negative-Strand RNA

2001 ◽  
Vol 75 (4) ◽  
pp. 1708-1721 ◽  
Author(s):  
Rajeev Banerjee ◽  
Asim Dasgupta
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Specific Interaction ◽  
Helicase Domain ◽  
Loop Structure ◽  
Stem Loop ◽  
Negative Strand ◽  
Stem Loop Structure ◽  
Rna Interaction ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and ΔpNS3 (helicase domain only) and examined their abilities to interact with the 3′-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3′ untranslated region (UTR). The results presented here demonstrate that NS3 (and ΔpNS3) interacts efficiently and specifically with the 3′-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5′-terminal RNA sequences. The interaction of NS3 with the 3′-terminal negative strand [called 3′(−) UTR127] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3′ terminus (nucleotides 5 to 20 from the 3′ end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and ΔpNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3′-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3′(−) UTR binding, specific interaction of NS3 (or ΔpNS3) with the 3′-terminal sequences of the positive-strand RNA [3′(+) UTR] appears to require the entire 3′(+) UTR of HCV. Deletion of either the 98-nucleotide 3′-terminal conserved region or the 5′ half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3′-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.

scholarly journals Structural and functional analysis of the roles of the HCV 5′ NCR miR122-dependent long-range association and SLVI in genome translation and replication

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5870
Author(s):  
Kirsten Bentley ◽  
Jonathan P. Cook ◽  
Andrew K. Tuplin ◽  
David J. Evans
Keyword(s):  
Long Range ◽  
Conformational Changes ◽  
Core Protein ◽  
Genome Replication ◽  
Translation Efficiency ◽  
Coding Region ◽  
Stem Loop ◽  
Protein Coding ◽  
Virus Rna ◽  
Rna Interaction

The hepatitis C virus RNA genome possesses a variety of conserved structural elements, in both coding and non-coding regions, that are important for viral replication. These elements are known or predicted to modulate key life cycle events, such as translation and genome replication, some involving conformational changes induced by long-range RNA–RNA interactions. One such element is SLVI, a stem-loop (SL) structure located towards the 5′ end of the core protein-coding region. This element forms an alternative RNA–RNA interaction with complementary sequences in the 5′ untranslated regions that are independently involved in the binding of the cellular microRNA 122 (miR122). The switch between ‘open’ and ‘closed’ structures involving SLVI has previously been proposed to modulate translation, with lower translation efficiency associated with the ‘closed’ conformation. In the current study, we have used selective 2′-hydroxyl acylation analysed by primer extension to validate this RNA–RNA interaction in the absence and presence of miR122. We show that the long-range association (LRA) only forms in the absence of miR122, or otherwise requires the blocking of miR122 binding combined with substantial disruption of SLVI. Using site-directed mutations introduced to promote open or closed conformations of the LRA we demonstrate no correlation between the conformation and the translation phenotype. In addition, we observed no influence on virus replication compared to unmodified genomes. The presence of SLVI is well-documented to suppress translation, but these studies demonstrate that this is not due to its contribution to the LRA. We conclude that, although there are roles for SLVI in translation, the LRA is not a riboswitch regulating the translation and replication phenotypes of the virus.

scholarly journals MicroRNA-135a Modulates Hepatitis C Virus Genome Replication through Downregulation of Host Antiviral Factors

2018 ◽  
Vol 34 (2) ◽  
pp. 197-210 ◽  
Author(s):  
Catherine Sodroski ◽  
Brianna Lowey ◽  
Laura Hertz ◽  
T. Jake Liang ◽  
Qisheng Li
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Genome ◽  
Genome Replication

scholarly journals Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

2009 ◽  
Vol 83 (10) ◽  
pp. 5137-5147 ◽  
Author(s):  
Hiromichi Hara ◽  
Hideki Aizaki ◽  
Mami Matsuda ◽  
Fumiko Shinkai-Ouchi ◽  
Yasushi Inoue ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Genome ◽  
Proteome Analysis ◽  
Virus Genome ◽  
Genome Replication ◽  
Creatine Kinase B ◽  
Efficient Replication

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.

scholarly journals Evaluation of Canonical siRNA and Dicer Substrate RNA for Inhibition of Hepatitis C Virus Genome Replication – A Comparative Study

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0117742 ◽  
Author(s):  
Bruno Carneiro ◽  
Ana Cláudia Silva Braga ◽  
Mariana Nogueira Batista ◽  
Mark Harris ◽  
Paula Rahal
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Comparative Study ◽  
Virus Genome ◽  
Genome Replication

Enzymatic properties of hepatitis C virus NS3-associated helicase

Microbiology ◽  
2000 ◽  
Vol 81 (5) ◽  
pp. 1335-1345 ◽  
Author(s):  
Chantal Paolini ◽  
Raffaele De Francesco ◽  
Paola Gallinari
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Structure ◽  
Structural Protein ◽  
Single Cycle ◽  
Detailed Characterization ◽  
Stem Loop ◽  
Hybrid Substrates ◽  
Serine Protease Activity

The hepatitis C virus non-structural protein 3 (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. In this study, an N-terminal hexahistidine-tagged full-length NS3 polypeptide was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. Detailed characterization of the helicase activity of NS3 is presented with regard to its binding and strand release activities on different RNA substrates. On RNA double-hybrid substrates, the enzyme was shown to perform unwinding activity starting from an internal ssRNA region of at least 3 nt and moving along the duplex in a 3′ to 5′ direction. In addition, data are presented suggesting that binding to ATP reduces the affinity of NS3 for ssRNA and increases its affinity for duplex RNA. Furthermore, we have ascertained the capacity of NS3 to specifically interact with and resolve the stem–loop RNA structure (SL I) within the 3′-terminal 46 bases of the viral genome. Finally, our analysis of NS3 processive unwinding under single cycle conditions by addition of heparin in both helicase and RNA-stimulated ATPase assays led to two conclusions: (i) NS3-associated helicase acts processively; (ii) most of the NS3 RNA-stimulated ATPase activity may not be directly coupled to translocation of the enzyme along the substrate RNA molecule.

Sign in / Sign up

Export Citation Format

Share Document