Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication

Evans, Matthew J.; Rice, Charles M.; Goff, Stephen P.

doi:10.1073/pnas.0405152101

Cited by 289 publications

(305 citation statements)

References 40 publications

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“…As shown in Figure 3, all the HCV nonstructural proteins studied, including NS3, NS4B, NS5A and NS5B, were found in the DRM fraction. hVAP-33, which is involved in the formation of HCV RC [8,37], was also detected in the DRM. Significantly, PTB was detected in this membrane fraction in the replicon-containing cells, but not in Huh-7 cells.…”

Section: Ptb Localized In Membrane Fractions Of Replicon Cellsmentioning

confidence: 91%

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

et al. 2006

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The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane proteinassociated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.

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Section: Ptb Localized In Membrane Fractions Of Replicon Cellsmentioning

confidence: 91%

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

et al. 2006

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“…It is implicated in directing NS proteins to cholesterol-rich, detergent-resistant membranes on which RNA replication is thought to occur (59). Interaction between VAP-A and NS5A is enhanced by cell culture-adaptive mutations suggesting that hyperphosphorylation of NS5A negatively affects association with this host factor (44). More recently, VAP-B, an isoform of VAP-A, was found to interact with NS5A and NS5B, too (60).…”

Section: Components Of the Hcv Replication Complexmentioning

confidence: 99%

“…However, phosphorylation of NS5A is a conserved feature among hepaci-and pestiviruses and also found with flavivirus NS5, arguing that phosphorylation plays an important role in the HCV life cycle (43). Moreover, it was found that mutations reducing NS5A hyperphosphorylation can lead to a dramatic enhancement of viral RNA replication (36,44). 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.…”

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

176

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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“…So far, it has been shown only for NS5A that adaptive mutations alter the phosphorylation state of the NS5A protein, thereby affecting the interaction with the host protein VAP-A (vesicle-associated membrane protein-associated protein A). 21 HCV replicons have proven to be extremely valuable for studies on the process of HCV replication, as well as for testing of novel antiviral compounds that specifically target the protease activity of NS3 or the polymerase activity of NS5. 22 This has been further facilitated by the creation of subgenomic replicons containing reporter genes such as firefly luciferase or fluorescent proteins, which allows their use in high-throughput screening assays to determine the efficacy of replication inhibitors.…”

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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Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication

Cited by 289 publications

References 40 publications

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

Polypyrimidine-tract-binding Protein is a Component of the HCV RNA Replication Complex and Necessary for RNA Synthesis

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

Experimental models for hepatitis C viral infection

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