The Hepatitis C Virus NS4B Protein Can
            <i>trans</i>
            -Complement Viral RNA Replication and Modulates Production of Infectious Virus

Jones, Daniel M.; Patel, Arvind H.; Targett-Adams, Paul; McLauchlan, John

doi:10.1128/jvi.01885-08

Cited by 125 publications

(162 citation statements)

References 68 publications

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“…This implication is consistent with an earlier report suggesting that disrupting the amphipathic N-terminal helix of NS4B by site-directed mutagenesis abolished HCV RNA replication in a subgenomic replicon system from HCV genotype 1b ( 9 ). In addition, other studies demonstrated that residues in the C-terminus of HCV NS4B in HCV genotype 2a subgenomic replicon were critical for viral RNA replication ( 10 ). The same group of investigators suggested that the C-terminal domain of NS4B infl uences production of infectious virus.…”

supporting

confidence: 81%

Association of lipid droplet and hepatitis C virus proteins: insights for virus replication

Bose

Ray

2013

Journal of Lipid Research

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Hepatitis C virus (HCV) infection represents a major worldwide disease burden, with an estimated prevalence of >185 million people ( 1 ). Among the various comorbidities associated with chronic HCV infection, hepatic steatosis is a frequent complication, and its presence is a key prognostic indicator associated with progression to hepatic fi brosis and with the development of hepatocellular carcinoma ( 2 ). Several mechanisms have been proposed to account for the development of steatosis and fatty liver in the setting of HCV infection ( 3 ). HCV infection enhances lipogenesis, reduces secretion of VLDL, attenuates ␤ -oxidation of lipids, and increases virus growth and replication through complex pathways that intersect via modulating host cell lipid metabolism ( 4 ). In addition, recent studies have implicated lipid droplet formation and turnover in the life cycle of HCV. Specifi cally, studies have identifi ed a role for the triglyceride-synthesizing enzyme diacylglycerol acyltransferase 1 (DGAT1) in both the formation of lipid droplets within the bilayer of the endoplasmic reticulum (ER) and also in the formation of infectious virions ( 5 ). Those studies demonstrated that DGAT1 (but not DGAT2) physically interacts with components of the nucleocapsid core and in turn recruits the viral replication complex to the newly generated lipid droplets ( 5 ). In addition, more-recent studies have demonstrated that this physical interaction between DGAT1 and HCV core components then interferes with triglyceride lipolysis and lipid droplet turnover, an effect that further enhances hepatic steatosis ( 6 ). These fi ndings collectively suggest that HCV infection impacts hepatic lipid metabolism over a broad range of pathways, including coopting elements of lipid droplet formation and turnover as well as modulating lipogenesis, FA oxidation, and VLDL secretion. However, despite the obvious public health impact and importance of HCV infection and its role in modulating hepatic lipid metabolism, there is still much to learn about the molecular and biochemical mechanisms by which the structural and nonstructural protein components of the HCV function within these pathways. This relative dearth of information is at least partially explained by the lack of preclinical experimental models of replicating HCV. Accordingly, most of the available information still derives from studies using transfected cell lines that support HCV genome replication, either as subgenomic or full-length constructs.With this background, the article from Tanaka et al. ( 7 ) in this issue of The Journal of Lipid Research provides new insights into the role of HCV NS4B (a nonstructural protein of the virus, known to be an integral ER membrane protein) and its association with lipid droplets. The authors demonstrated this association using state-of-the-art confocal imaging and biochemical fractionation of cell lines supporting HCV replication. Although the association of HCV with lipid droplets is well known (Moradpour et al., 1996; Miyanari et al., 2007, c...

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supporting

confidence: 81%

Association of lipid droplet and hepatitis C virus proteins: insights for virus replication

Bose

Ray

2013

Journal of Lipid Research

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“…The hypo-(black arrowhead) and hyperphosphorylated (grey arrowhead) species of NS5A are indicated and actin detection served as a loading control. cells were transfected with siRNAs against each gene and then infected with HCV virions generated by J6-JFH1, a chimeric construct that gives virus titres, which are about 10-fold greater than strain JFH1 (Jones et al, 2009). At 48 h after infection (i.e.…”

Section: Isolation Of Cell Lines Supporting Autonomous Replication Ofmentioning

confidence: 99%

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

Jones¹,

Domingues²,

Targett-Adams³

et al. 2010

Journal of General Virology

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Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 59-39 exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins. INTRODUCTIONHepatitis C virus (HCV) possesses a single-stranded (ss), positive-sense RNA genome of approximately 9.6 kb . The RNA is translated to yield a polyprotein that is cleaved by cellular (signal peptidase and signal peptide peptidase) and virus-encoded proteases (the NS2-3 autoprotease and NS3-4A serine protease), thereby generating the mature HCV proteins. The nonstructural (NS) proteins NS3, NS4A, NS4B, NS5A and NS5B are located at punctate structures on the endoplasmic reticulum (ER) membrane, which represent viral replication complexes (RCs) (Egger et al., 2002;Gosert et al., 2003;Targett-Adams et al., 2008). Although the NS3-NS5B proteins are essential for HCV RNA synthesis, replication is dependent on host cell factors. A number of cellular proteins that participate in HCV RNA replication have been identified (see below for references). However, characterization of the complete complement of cellular factors required for HCV replication remains undefined.Small inhibitory RNA (siRNA) technology is a powerful approach to identify host cell factors that contribute to viral replication and as such, several groups have screened siRNA libraries targeting cellular genes. For example, screening with libraries that target protein kinases (Supekova et al., 2008), transporter proteins and transcription factors (Ng et al., 2007), previously documented HCV-interacting proteins (Randall et al., 2007) and proteins implicated...

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“…In fact most of the Con1-replication enhancing changes within the viral non-structural proteins stimu lated replication at the expense of production of infectious particles. This observation was a first hint suggesting that the non-structural proteins contribute to production of infectious HCV, an observation which was much refined and extended using the infectious JFH1 system (Jones, Murray et al 2007;Steinmann, Penin et al 2007;Yi, Ma et al 2007;Appel, Zayas et al 2008;Ma, Yates et al 2008;Jones, Patel et al 2009;Phan, Beran et al 2009). Besides this, the observation that REMs can interfere with virus production in Con1 genomes provided a simple explanation why the adapted Con1 gen omes unlike the wild type were non-infectious in Chimpanzee or re verted back to the wild type sequence .…”

Section: Cell Culture Infectious Hcv Genomes and Host Cellsmentioning

confidence: 99%

Cell Culture Systems for Hepatitis C Virus

Steinmann

Pietschmann

2013

Current Topics in Microbiology and Immunology

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Due to the obligatory intracellular life style of viruses, cell culture systems for efficient viral propagation are crucial to obtain a detailed understanding of the virus host cell interaction. For hepatitis C virus (HCV) the development of permissive and authentic culture models has been and continues to be a challenging task. The first ef forts to culture HCV had limited success and range back to before the virus was molecularly cloned in 1989. Since then several major breakthroughs have gradually overcome limitations in culturing the virus and sequentially permitted analysis of viral RNA replication, cell entry and ultimately the complete replication cycle in cultured cells in 2005. Until today, basic and applied HCV research greatly benefit from these tremendous efforts which spurred multiple com plementary cell based model systems for distinct steps of the HCV replication cycle. When used in combination they now permit deep insights into the fascinating biology of HCV and its interplay with the host cell. In fact drug development has been much facilitated and our understanding of the molecular determinants of HCV replication has grown in parallel to these advances. Building on this ground work and further refining our cellular models to better mimic the ar chitecture, polarization and differentiation of natural hepatocytes should reveal novel unique aspects of HCV replication. Ultimately, models to culture primary HCV isolates across all genotypes may teach us important new lessons about viral functional adaptations that have evolved in exchange with its human host and that may ex plain the variable natural course of hepatitis C.

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The Hepatitis C Virus NS4B Protein Can trans -Complement Viral RNA Replication and Modulates Production of Infectious Virus

Cited by 125 publications

References 68 publications

Association of lipid droplet and hepatitis C virus proteins: insights for virus replication

Association of lipid droplet and hepatitis C virus proteins: insights for virus replication

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

Cell Culture Systems for Hepatitis C Virus

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