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

Friebe, Peter; Bartenschlager, Ralf

doi:10.1128/jvi.76.11.5326-5338.2002

Cited by 249 publications

(266 citation statements)

References 64 publications

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“…As this overlaps with the IRES, there is considerable interest in understanding how this region might modulate translation and replication, which are unlikely to occur simultaneously on the same RNA template. The 3፱ NCR has been found to contain a nonessential variable region, a poly-U/UC tract that must be more than 26 nucleotides long, followed by a highly conserved and essential 3፱X domain 55,57,76 . Recently a conserved stem-loop structure within the NS5B coding region, 5BSL3.2, was found to be required for RNA replication 78 .…”

Section: Mechanisms Of Hcv Rna Replicationmentioning

confidence: 99%

Unravelling hepatitis C virus replication from genome to function

Lindenbach

Rice

2005

Nature

753

561

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Since the discovery of the hepatitis C virus over 15 years ago, scientists have raced to develop diagnostics, study the virus and find new therapies. Yet virtually every attempt to dissect this pathogen has met with roadblocks that impeded progress. Its replication was restricted to humans or experimentally infected chimpanzees, and efficient growth of the virus in cell culture failed until very recently. Nevertheless hard-fought progress has been made and the first wave of antiviral drugs is entering clinical trials.virus life cycle: first, as a messenger RNA (mRNA) for translation of the viral proteins; second as a template for RNA replication; and third, as a nascent genome packaged within new virus particles. Virions presumably form by budding into the endoplasmic reticulum (ER) and leave the cell through the secretory pathway.Researchers have followed each aspect of the virus life cycle in turn. Just as infection starts from an HCV genome entering the cytoplasm and progressing through translation, replication and particle production, our understanding has progressed from having a genome sequence to understanding translation and the viral gene products, characterizing RNA replication, and establishing systems to characterize virus particles and infectivity. In this review, we summarize the current understanding of HCV replication with special emphasis on recent developments. As space is limited, we cannot be comprehensive; readers may wish to consult other reviews for detail and breadth 5,13,14. Initial studies: HCV translation and polyprotein processingThe identification of HCV yielded a partial viral genome sequence 5 . Research in the early 1990s focused on dissecting HCV gene expression and characterizing the gene products. Much has been learned about the biochemistry of three key enzymes, and structural information is now available at the atomic level for roughly half of the proteincoding region.Translation of the HCV genome, which lacks a 5፱ cap, depends on an internal ribosome entry site (IRES) within the 5፱-noncoding region (NCR). The HCV IRES binds 40S ribosomal subunits directly and avidly, bypassing the need for pre-initiation factors, and inducing an mRNA-bound conformation in the 40S subunit 15 . The IRES-40S complex then recruits eukaryotic initiation factor (eIF) 3 and the ternary complex of Met-tRNA-eIF2-GTP to form a non-canonical 48S intermediate, before a kinetically slow transition to the translationally active 80S complex 16,17 .Once initiated, translation of the HCV genome produces a large polyprotein that is proteolytically cleaved to produce 10 viral proteins (Fig. 2a). The amino-terminal one-third of the polyprotein encodes the virion structural proteins: the highly basic core (C) protein, and glycoproteins E1 and E2. After the structural region comes a small integral membrane protein, p7, which seems to function as an ion channel 18,19 . The remainder of the genome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which coordinate the intracellular process...

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Section: Mechanisms Of Hcv Rna Replicationmentioning

confidence: 99%

Unravelling hepatitis C virus replication from genome to function

Lindenbach

Rice

2005

Nature

753

561

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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.

468

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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“…Mapping of cis-acting HCV RNA required for RNA synthesis both in vitro and in vivo indicated that the X-RNA is a minimal cis-acting RNA essential for HCV minus-strand RNA synthesis (Oh et al, 2000;Friebe and Bartenschlager, 2002). Furthermore, synthesis of minus-strand HCV RNA was shown to be initiated from the internal region of X-RNA with N-terminal (His)6 tagged full-length NS5B and E. coli lysyl-tRNA synthetase-fused NS5B (Oh et al, 2000;Kim et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

Choi

Kim

Oh³

2003

Exp Mol Med

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A bstractThe hepatitis C virus (HCV) RNA-dependent RNA polym erase, NS5B protein, is the key viral enzym e responsible for replication of the HCV viral RNA genom e. Although several full-length and truncated form s of the HCV NS5B proteins have been expressed previously in insect cells, contam ination of host term inal transferase (TNTase) has ham pered analysis of the RNA synthesis initiation m echanism using natural HCV RNA tem plates. W e have expressed the HCV NS5B protein in insect cells using a recom binant baculovirus and purified it to near hom ogeneity w ithout contam inated TNTase. The highly purified recom binant HCV NS5B w as capable of copying 9.6-kb full-length HCV RNA tem plate, and m ini-HCV RNA carrying both 5'-and 3'-untranslated regions (UTRs) of the HCV genom e. In the absence of a prim er, and other cellular and viral factors, the NS5B could elongate over HCV RNA tem plates, but the synthesized products were prim arily in the double stranded form , indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA tem plates representing the 3'-end region of HCV m inus-strand RNA and the X-RNA at the 3'-end of HCV RNA genom e w as also initiated de novo. N o form ation of dim er-size self-prim ed RNA products resulting from extension of the 3'-end hydroxyl group w as observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polym erase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genom e.

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

Cited by 249 publications

References 64 publications

Unravelling hepatitis C virus replication from genome to function

Unravelling hepatitis C virus replication from genome to function

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

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

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