Cyclosporine A inhibits hepatitis C virus nonstructural protein 2 through cyclophilin A #

Ciesek, Sandra; Steinmann, Eike; Wedemeyer, Heiner; Manns, Michael P.; Neyts, Johan; Tautz, Norbert; Madan, Vanesa; Bartenschlager, Ralf; Hahn, Thomas von; Pietschmann, Thomas

doi:10.1002/hep.23281

Cited by 107 publications

(85 citation statements)

References 23 publications

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“…This suggests that SCD1 may be involved in an additional step as well as a replication step of the viral life cycle. Indeed, it has been reported previously that cyclophilin A inhibitors target multiple steps of the HCV life cycle and inhibit JFH1-derived full-length HCV more efficiently than subgenomic replicons (41,42). We also confirmed that SCD1 was not involved in HCV IRES-mediated translation.…”

Section: Discussionsupporting

confidence: 79%

Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication

Nguyen

Lim

Pham

et al. 2014

J Virol

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The hepatitis C virus (HCV) life cycle is tightly regulated by lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have recently screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet formation using cell culture-grown HCV (HCVcc)-infected cells. We selected and characterized the gene encoding stearoyl coenzyme A (CoA) desaturase 1 (SCD1). siRNA-mediated knockdown or pharmacological inhibition of SCD1 abrogated HCV replication in both subgenomic replicon and Jc1-infected cells, while exogenous supplementation of either oleate or palmitoleate, products of SCD1 activity, resurrected HCV replication in SCD1 knockdown cells. SCD1 was coimmunoprecipitated with HCV nonstructural proteins and colocalized with both double-stranded RNA (dsRNA) and HCV nonstructural proteins, indicating that SCD1 is associated with HCV replication complex. Moreover, SCD1 was fractionated and enriched with HCV nonstructural proteins at detergent-resistant membrane. Electron microscopy data showed that SCD1 is required for NS4B-mediated intracellular membrane rearrangement. These data further support the idea that SCD1 is associated with HCV replication complex and that its products may contribute to the proper formation and maintenance of membranous web structures in HCV replication complex. Collectively, these data suggest that manipulation of SCD1 activity may represent a novel host-targeted antiviral strategy for the treatment of HCV infection. IMPORTANCEStearoyl coenzyme A (CoA) desaturase 1 (SCD1), a liver-specific enzyme, regulates hepatitis C virus (HCV) replication through its enzyme activity. HCV nonstructural proteins are associated with SCD1 at detergent-resistant membranes, and SCD1 is enriched on the lipid raft by HCV infection. Therein, SCD1 supports NS4B-mediated membrane rearrangement to provide a suitable microenvironment for HCV replication. We demonstrated that either genetic or chemical knockdown of SCD1 abrogated HCV replication in both replicon cells and HCV-infected cells. These findings provide novel mechanistic insights into the roles of SCD1 in HCV replication. Hepatitis C virus (HCV) is an enveloped virus with a positivesense, single-stranded RNA virus that belongs to the genus Hepacivirus in the family Flaviviridae (1). Approximately 170 million people are chronically infected with HCV worldwide. Three million people are newly infected with HCV annually, and more than 350,000 individuals die from HCV-related liver diseases every year (1, 2). Current standard therapy elicits some side effects and results in a sustained virological response in only certain genotypes of HCV patients. Recently, the U.S. Food and Drug Administration approved various direct-acting antivirals (DAAs), including boceprevir, telaprevir, sofosbuvir, and simeprevir, for triple therapy in combination with pegylated interferon and ribavirin for patients with certain genotypes. Nevertheless, these new DAAs also have had some lim...

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Section: Discussionsupporting

confidence: 79%

Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication

Nguyen

Lim

Pham

et al. 2014

J Virol

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“…The power of this method is illustrated by NS5A and CypA. These two proteins are involved in HCV assembly (57,58); they cosediment with virions in density gradients (Figs. 1A and 2E), but they are not detectable in virus fractions after affinity purification (Fig.…”

Section: Discussionmentioning

confidence: 99%

Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome

Merz

Long

Hiet

et al. 2011

Journal of Biological Chemistry

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313

355

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A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was Nterminally tagged with a FLAG epitope. This virus, designated Jc1E2 FLAG , produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2 FLAG particles to resemble the one very low-and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2 FLAG particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.

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“…Recent studies also suggested that NS2, a cysteine protease, is an additional cyclophilin target (19,25). NS5A mutations, mostly located in the C-terminal half of the protein, were shown to confer the highest resistance against cyclophilin inhibitors (21,22).…”

mentioning

confidence: 99%

Domain 3 of NS5A Protein from the Hepatitis C Virus Has Intrinsic α-Helical Propensity and Is a Substrate of Cyclophilin A

Verdegem

Badillo

Wieruszeski

et al. 2011

Journal of Biological Chemistry

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101

106

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Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an ␣-helix. NMR analysis identifies two putative ␣-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA. Hepatitis C virus (HCV),3 a small enveloped virus from the Flaviviridae family, is a major cause of chronic liver disease that may lead to steatosis, liver cirrhosis, and hepatocellular carcinoma. Given about 180 million chronically infected individuals worldwide, HCV is an important health challenge (1). Current therapy is based on a combination of pegylated interferon-␣ and ribavirin but is not fully satisfying because numerous patients are not responding or suffer from serious side effects caused by this treatment. The development of new drugs to treat HCV infections requires a better understanding of the structural and functional features of the viral proteins and their relationships with host cell factors. The HCV genome (ϳ9.6 kb) encodes for a single polyprotein precursor (ϳ3,000 aa) that is co-and post-translationally processed by cellular and viral proteases to yield 10 mature proteins (2, 3). They are classified into structural proteins (Core, E1, and E2), which constitute the viral particle, and nonstructural proteins, of which two, p7 and nonstructural protein 2 (NS2), are required for virus assembly. The remainder of the nonstructural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) are involved in HCV RNA replication (reviewed in Refs. 2 and 3).Cyclophilins are host cell factors that in addition to viral proteins, are equally essential for HCV replication. The human genome encodes up to 16 different cyclophilins (4) that, despite differences in their tissue distributio...

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Cyclosporine A inhibits hepatitis C virus nonstructural protein 2 through cyclophilin A #

Cited by 107 publications

References 23 publications

Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication

Stearoyl Coenzyme A Desaturase 1 Is Associated with Hepatitis C Virus Replication Complex and Regulates Viral Replication

Biochemical and Morphological Properties of Hepatitis C Virus Particles and Determination of Their Lipidome

Domain 3 of NS5A Protein from the Hepatitis C Virus Has Intrinsic α-Helical Propensity and Is a Substrate of Cyclophilin A

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