3′ Nontranslated RNA Signals Required for Replication of Hepatitis C Virus RNA

Yi, Min Kyung; Lemon, Stanley M.

doi:10.1128/jvi.77.6.3557-3568.2003

Cited by 186 publications

(193 citation statements)

References 52 publications

(84 reference statements)

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“…3). It has been reported that the length and composition of the poly(U/UC) tract is important for the replication of HCV replicons (Friebe & Bartenschlager, 2002;Yi & Lemon, 2003;You & Rice, 2008). However, no replication advantage of a poly(U/UC) tract longer than 86 bp was revealed in this study.…”

Section: Discussioncontrasting

confidence: 41%

“…Each segment of HCV was cloned into this vector to generate the full-length clones. The HCV-KT9 clone was established using the 39-terminal fragment with the longest poly(U/UC) tract length (115 nt), which should have a high replication ability (Friebe & Bartenschlager, 2002;Yi & Lemon, 2003;You & Rice, 2008). A clone with a shorter poly(U/UC) tract length (86 nt), HCV-KT1, was also generated.…”

Section: Methodsmentioning

confidence: 99%

“…We obtained 24 39-extremity clones with different poly(U/UC) tract lengths. We selected the clone with the longest (U/UC) tract because a previous study indicated that the length of poly(U/UC) tract correlates with HCV replication in an HCV replicon system (Friebe & Bartenschlager, 2002;Yi & Lemon, 2003;You & Rice, 2008). The length of the poly(U/UC) tract in the longest 39 clone was 115 nt.…”

Section: Characteristics Of Genotype 1b Clones Hcv-kt9 and Hcv-kt1mentioning

confidence: 99%

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Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice

Kimura

Imamura

Hiraga

et al. 2008

Journal of General Virology

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The establishment of clonal infection of hepatitis C virus (HCV) in a small-animal model is important for the analysis of HCV virology. A previous study developed models of molecularly cloned genotype 1a and 2a HCV infection using human hepatocyte-transplanted chimeric mice. This study developed a new model of molecularly cloned genotype 1b HCV infection. A full-length genotype 1b HCV genome, HCV-KT9, was cloned from a serum sample from a patient with severe acute hepatitis. The chimeric mice were inoculated intrahepatically with in vitro-transcribed HCV-KT9 RNA. Inoculated mice developed viraemia at 2 weeks post-infection, and this persisted for more than 6 weeks. Passage experiments indicated that the sera of these mice contained infectious HCV. Interestingly, a similar clone, HCV-KT1, in which the poly(U/UC) tract was 29 nt shorter than in HCV-KT9, showed poorer in vivo infectivity and replication ability. An in vitro study showed that no virus was produced in the culture medium from HCV-KT9-transfected cells. In conclusion, this study developed a genetically engineered genotype 1b HCV-infected mouse. This mouse model will be useful for the study of HCV virology, particularly the mechanism underlying the variable resistance of HCV genotypes to interferon therapy.

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Section: Discussioncontrasting

confidence: 41%

Section: Methodsmentioning

confidence: 99%

Section: Characteristics Of Genotype 1b Clones Hcv-kt9 and Hcv-kt1mentioning

confidence: 99%

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Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice

Kimura

Imamura

Hiraga

et al. 2008

Journal of General Virology

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“…The HCV polyU/UC region is a flexible motif among HCV strains and is essential for viral replication 13 . In the HCV genotype 1b strain used in these experiments, the poly-U/UC region is comprised of 100 nt containing 78% uridine and 22% ribocytosine (Fig.…”

mentioning

confidence: 99%

Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

Saito

Owen

Jiang

et al. 2008

Nature

661

735

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Innate immune defenses are essential for the control of virus infection and are triggered through host recognition of viral macromolecular motifs known as pathogen-associated molecular patterns (PAMPs) 1. Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people 2. Infection is governed by hepatic immune defenses triggered by the cellular RIG-I helicase. RIG-I binds PAMP RNA and signals IRF-3 activation to induce the expression of α/β interferon (IFN) and antiviral/interferon-stimulated genes (ISGs) that limit infection 3 -10. Here we identified the poly-uridine motif of the HCV genome 3' nontranslated region (NTR) as the PAMP substrate of RIG-I, and show that this and similar homopoly-uridine motifs present in the genome of RNA viruses is the chief feature of RIG-I recognition and immune triggering 8. 5' terminal triphosphate on the PAMP RNA was necessary but not sufficient for RIG-I binding, which was primarily dependent upon homopolymeric ribonucleotide composition, linear structure and length. The HCV PAMP RNA stimulated RIG-I-dependent signaling to induce a hepatic innate immune response in vivo, and triggered IFN and ISG expression to suppress HCV infection in vitro. These results provide a conceptual advance by identifying homopoly-uridine motfis present in the genome of HCV and other RNA viruses as the PAMP substrate of RIG-I, and define immunogenic features of the PAMP/RIG-I interaction that could be utilized as an immune adjuvant for vaccine and immunotherapy approaches.To determine the nature of the HCV PAMP RNA we conducted a functional screen to identify possible HCV PAMP RNA motifs. We assessed the ability of full length HCV genome RNA or contiguous HCV RNA segments to trigger the IFN-β promoter in transfected Huh7 cells. The full-length HCV genome RNA triggered innate immune signaling to induce the IFN-β promoter (Fig. 1a). Two regions of the HCV RNA, encoding nt 2406-3256 and nt 8872-9616, stimulated significant induction of the IFN-β promoter (Fig. 1b) with signaling activity respectively localized to nt 2406-2696 of the open reading frame and nt 9389-9619 encoding the 3' NTR (Fig.1c). Deletion of nt 9389-9619 but not nt 2408-2663 from the HCV genome significantly attenuated signaling to the IFN-β promoter (Fig 1d). PAMP motifs are typically conserved among strains of a pathogen 1 , and sequence comparison of multiple HCV genomes revealed global variability within nt 2406-3696 among virus strains but nt 9389-9616 encoded motifs of high conservation (Fig. S1) Thus, the viral 3' NTR might encode HCV PAMP motifs that trigger innate immune signaling in the host cell.The HCV 3' NTR is comprised of three regions: a variable region (VR) with potential secondary structure, a nonstructured poly-U/UC region containing polyuridine with interspersed ribocytidine, and the terminal X region containing three conserved stem-loop structures (Fig. 1e) 12 . We evaluated the ability of RNA encoding the HCV 3' NTR or each of its regions to trigger intracellular si...

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“…The HCV genome is a positivestranded Ϸ9.6-kb RNA molecule consisting of a single ORF, which is flanked by 5Ј and 3Ј untranslated regions (UTR). The HCV 5Ј UTR contains a highly structured internal ribosome entry site (3)(4)(5)(6)(7)(8), and the 3Ј UTR is essential for replication (9,10). The HCV ORF encodes a single 3,008-to 3,037-aa polyprotein that is posttranslationally processed to produce Ն10 different proteins: core protein, envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1,8,11).…”

mentioning

confidence: 99%

Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids

Kapadia

Chisari²

2005

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

467

413

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Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Our laboratory has previously demonstrated that high-level HCV replication during acute infection of chimpanzees is associated with the modulation of multiple genes involved in lipid metabolism, and that drugs that regulate cholesterol and fatty acid biosynthesis regulate the replication of the subgenomic HCV replicon in Huh-7 cells. In this article, we demonstrate that Huh-7 cells harboring replicating, full-length HCV RNAs express elevated levels of ATP citrate lyase and acetyl-CoA synthetase genes, both of which are involved in cholesterol and fatty acid biosynthesis. Further, we confirm that the cholesterol-biosynthetic pathway controls HCV RNA replication by regulating the cellular levels of geranylgeranyl pyrophosphate, we demonstrate that the impact of geranylgeranylation depends on the fatty acid content of the cell, and we show that fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation. These results illustrate a complex cellular-regulatory network that controls HCV RNA replication, presumably by modulating the trafficking and association of cellular and͞or viral proteins with cellular membranes, suggesting that pharmacologic manipulation of these pathways may have a therapeutic effect in chronic HCV infection.cholesterol ͉ replicon H epatitis C virus (HCV), a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis and hepatocellular carcinoma (1, 2). The HCV genome is a positivestranded Ϸ9.6-kb RNA molecule consisting of a single ORF, which is flanked by 5Ј and 3Ј untranslated regions (UTR). The HCV 5Ј UTR contains a highly structured internal ribosome entry site (3-8), and the 3Ј UTR is essential for replication (9, 10). The HCV ORF encodes a single 3,008-to 3,037-aa polyprotein that is posttranslationally processed to produce Ն10 different proteins: core protein, envelope proteins E1 and E2, p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1,8,11). Our understanding of the biology of HCV RNA replication has been greatly facilitated by the development of subgenomic and full-length HCV replicons that express HCV proteins and replicate their RNA in stably transfected human hepatoma-cell-derived Huh-7 cells.Our laboratory has previously demonstrated (12) that multiple cellular genes involved in lipid metabolism are differentially regulated during viral spread in acutely infected chimpanzees, and that ATP citrate lyase, which is required for cholesterol and fatty acid biosynthesis, is induced during the initial rise of high-level HCV replication during acute infection in chimpanzees. There is considerable evidence suggesting that the cholesterol and fatty-acid-biosynthesis pathways may play a role in HCV replication and infection. Steatosis, i.e., the formation of hepatocellular lipid droplets, is a well documented histological characteristic of HCV infection in humans, chimpanzees, and mouse m...

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3′ Nontranslated RNA Signals Required for Replication of Hepatitis C Virus RNA

Cited by 186 publications

References 52 publications

Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice

Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice

Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA

Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids

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