Efficient Initiation of HCV RNA Replication in Cell Culture

Blight, Keril J.; Kolykhalov, Alexander A.; Rice, Charles M.

doi:10.1126/science.290.5498.1972

Cited by 1,320 publications

(1,380 citation statements)

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“…By using transfection of the human hepatoma cell line Huh-7 with HCV replicons, it was found that these RNAs amplify only to low levels unless they acquire "cell culture adaptive" mutations. Many of these mutations cluster among others, in LCS I of NS5A, and they often reduce hyperphosphorylation arguing that this modification is of disadvantage for efficient RNA replication (8,45). In support of this assumption it was found that treatment of cells carrying non-adapted replicons with an inhibitor of the cellular kinase(s) responsible for NS5A hyperphosphorylation leads to an increase of RNA replication (46).…”

Section: Components Of the Hcv Replication Complexsupporting

confidence: 55%

“…However, some details begin to emerge due to the availability of efficient in vitro systems, most notably the HCV replicon system (7,8), HCV pseudoparticles (9, 10), and, very recently, the first system for efficient production of infectious HCV in cell culture (11)(12)(13). Since the minimum components required for HCV RNA translation and replication are the NTRs and the NS3 to NS5B coding region, in the following we will only refer to these viral elements.…”

Section: Components Of the Hcv Replication Complexmentioning

confidence: 99%

See 1 more Smart Citation

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Appel

Schaller

Pénin

et al. 2006

Journal of Biological Chemistry

177

120

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With an estimated number of about 180 million affected people worldwide and a limited therapy option, infection with the hepatitis C virus (HCV) 2 is an important medical problem. Initial limitations in propagating HCV in cell culture have been overcome step-by-step in the past few years and culminated in the recent development of a system allowing efficient propagation of infectious HCV in tissue culture. Nevertheless, structural and biochemical studies of viral proteins as well as molecular analysis of viral RNA replication are still major challenges. The recent resolution of the three-dimensional structures of some HCV proteins or domains along with elegant reverse genetic and cell biological studies provided first insights into how HCV amplifies its RNA, exploits the cellular machinery, and eventually overcomes innate immune responses. Organization of the Viral GenomeHCV is a positive strand RNA virus that has been classified as the genus Hepacivirus in the Flaviviridae family. The HCV genome is an uncapped, linear molecule with a length of ϳ9600 nucleotides (nt; Fig. 1A). It carries a long open reading frame that is flanked at the 5Ј-and 3Ј-ends by short highly structured non-translated regions (NTRs). The 5Ј-NTR has a length of about 340 nt and contains an internal ribosome entry site (IRES) required for translation of the HCV genome (1). This RNA element binds the 40 S ribosomal subunit in the absence of other translation initiation factors in a way that the initiation codon is placed in the immediate vicinity of the P site (2). Part of the IRES (domain II) overlaps with RNA signals essential for viral replication (Fig. 1A) arguing for a possible role of domain II in regulating a translation-RNA replication switch. The 3Ј-NTR has a tripartite structure composed of an ϳ40-nt-long variable region downstream of the HCV coding sequence, a poly(U/UC) tract of heterogeneous length, and a highly conserved 98-nt-long sequence designated X-tail (Fig. 1A). Genetic studies have shown that a poly(U/UC) tract of at least ϳ25 nt as well as the complete X-tail are required for RNA replication in cell culture and for infectivity of the viral genome in vivo. An additional cis-acting RNA element (CRE) has been identified in the 3Ј-terminal coding region of NS5B (Fig. 1A). This CRE (designated 5BSL3.2) (3) forms a long distance RNA-RNA interaction with SL2 in the X-tail (4), which is indispensable for RNA replication.Expression of the viral proteins from the monocistronic genome is primarily achieved by production of a polyprotein that is proteolytically cleaved into the structural proteins (core, envelope proteins E1 and E2), the hydrophobic peptide p7, and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Fig. 1A) (1, 5). Processing of the core to p7 region is mediated by host cell signalases, and in the case of the core protein, in addition by signal peptide peptidase. All remaining cleavages are carried out by two viral proteases: the NS2/3 protease mediating cleavage between NS2 and NS3 and the NS3...

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Section: Components Of the Hcv Replication Complexsupporting

confidence: 55%

Section: Components Of the Hcv Replication Complexmentioning

confidence: 99%

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Appel

Schaller

Pénin

et al. 2006

Journal of Biological Chemistry

177

120

View full text Add to dashboard Cite

show abstract

“…Introduction of these specific mutations in the wild-type consensus sequence significantly enhanced viral replication in vitro. [16][17][18][19][20] Mutational hot spots were found clustered primarily in the NS3, NS4B, and NS5A regions. The mechanisms behind the enhanced replication caused by these tissue-culture-adaptive mutations are still largely unknown, and the interesting fact that these mutations are not commonly found in patients suggests that these may have a toll on the viral fitness.…”

Section: Cell-based In Vitro Hcv Systemsmentioning

confidence: 99%

Experimental models for hepatitis C viral infection

Boonstra¹,

Laan²,

Vanwolleghem³

et al. 2009

Hepatology

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Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. The majority of infected individuals develop a persistent infection, which is associated with a high risk of liver cirrhosis and hepatocellular carcinoma. Since its discovery 20 years ago, progress in our understanding of this virus has been suboptimal due to the lack of good model systems. However, in the past decade this has greatly accelerated with the development of various in vitro cell culture systems and in vivo small-animal models. These systems have made a major impact on the field of HCV research, and have provided important breakthroughs in our understanding of HCV infection and replication. Importantly, the in vitro cell culture systems and the small-animal models have allowed preclinical testing of numerous novel antiviral compounds for the treatment of chronic HCV infection. In this article, we give an overview of current models, discuss their limitations, and provide future perspectives for research directed at the prevention and cure of hepatitis C. (HEPATOLOGY 2009;50:1646-1655.) Hepatitis C Virus Pathology and BiologyThe hepatitis C virus (HCV) is considered a successful pathogen in establishing persistent infections by evading the immune system. Only a fraction of acutely infected individuals are able to clear the infection spontaneously, whereas about 80% of the infected individuals develop a chronic infection. 1,2 The symptoms are initially mild in these persistently infected patients, and it may take decades before the serious consequences of chronic HCV infection become apparent. Patients with chronic HCV are at increased risk for developing liver fibrosis, cirrhosis, and/or hepatocellular carcinoma. Currently, these longterm complications of chronic HCV infection are the leading indication for liver transplantation. 3,4 Because of the high incidence of new infections by blood transfusions in the 1980s before discovery of the virus, and because morbidity associated with chronic HCV infections generally takes decades to develop, it is expected that the burden of disease in the near future will rise dramatically.HCV is an enveloped flavivirus, with a positivestranded RNA genome of approximately 9600 nucleotides and is composed of a single open reading frame, which encodes a polyprotein precursor of approximately 3000 amino acids. The coding region is flanked by 5Ј and 3Ј noncoding regions, which are important for the regulation of genomic duplication as well as initiation of translation. The single polyprotein is cleaved by host and viral proteases into individual structural and nonstructural (NS) proteins (Fig. 1A). Replication of the HCV genome involves the synthesis of a full-length negative-stranded RNA intermediate, which in turn provides a template for the de novo production of positive-stranded RNA. Both these synthesis steps are mediated by the viral RNA-dependent RNA polymerase NS5B. 5-7 NS5B lacks proofreading abilities, and this leads to a high mutation rate and the generation of numerous quasispecies. ...

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“…Subsequently, the Rice Lab identified several adaptive mutations in the HCV nonstructural protein NS5A. The new constructs conferred much increased replicative ability in vitro, as reported in 2000 (Blight et al, 2000). Continued search on HCV isolates with high in vitro replication efficiency yielded the most famous HCV cDNA clone, JFH-1 (genotype 2a).…”

mentioning

confidence: 95%