Inhibition of HCV Replication by Cyclophilin Antagonists Is Linked to Replication Fitness and Occurs by Inhibition of Membranous Web Formation

Madan, Vanesa; Paul, David; Lohmann, Volker; Bartenschlager, Ralf

doi:10.1053/j.gastro.2014.01.055

Cited by 68 publications

(83 citation statements)

References 47 publications

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“…Likewise, viral replication is also dependent on several host proteins, including cyclophilins (14,15), human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33) (16), phosphatidylinositol-4-kinase III alpha (PI4KIII-␣) (17), oxysterol-binding protein 1 (OSBP1) (18), NF90 and hnRNPL (19), etc. The importance of studying the role of these host proteins in the viral life cycle is underlined by the fact that many pharmaceutical agents directed against them, such as cyclophilin antagonists (20) and miR-122 inhibitors (21), are in clinical trials.…”

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confidence: 99%

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

Shwetha

Kumar

Mullick

et al. 2015

J Virol

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HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3= untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3= UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3= UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3= UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA.Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3= untranslated region (UTR) of HCV RNA. At the 3= UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions. Hepatitis C virus (HCV) was discovered in 1989 as a major cause of chronic non-A non-B hepatitis (1). HCV is an enveloped positive-strand RNA virus classified in the Hepacivirus genus within the Flaviviridae family. The single-stranded uncapped 9.6-kb RNA codes for a long polyprotein of ϳ3,000 amino acids which is then processed to structural and nonstructural proteins. The RNA genome is flanked by the 5= and 3= untranslated regions (UTRs), which are essential for translation and replication of the viral RNA. The viral proteins are synthesized by internal ribosome entry site (IRES)-mediated translation. These proteins initiate extensive remodeling of the intracellular membranes to create a detergent-resistant scaffold for viral replication, termed as the "membranous web" (2). The progeny viral genomes a...

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confidence: 99%

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

Shwetha

Kumar

Mullick

et al. 2015

J Virol

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“…Electron microscopy analyses suggest that NS5A-positive structures correspond, in part, to double membrane vesicles, which are a hallmark of the membranous HCV replication factory, designated membranous web (52). A NS5A "cluster" phenotype similar to that of the PW turn mutants was previously found in cells treated with NS5A or Cyp inhibitors (46,52,53), as well as in PI4KIII␣ knockdown cells. This phenotype was shown to correspond to lipid droplets or aberrant double membrane vesicles, respectively (47).…”

Section: Discussionmentioning

confidence: 73%

“…9B, right column). This altered phenotype correlated with a strong accumulation of large structures where NS5A is present, reminiscent of the cluster distribution in cells treated with NS5A and Cyp inhibitors (46,52,53). Notably, the proportion of cells with the cluster phenotype varied from 24 to 67% for I315G and P314A mutants, respectively, compared with 5% for WT (Fig.…”

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confidence: 77%

“…Therefore, inhibition of HCV replication, at least of the genotype 1b isolate Con1, by I315G and P314A mutations (Fig. 8) is probably due to disruption of the structural motif in NS5A-D2 and consequently to the abolishment of the functional interaction with CypA that is required for the establishment of HCV replication sites (46).…”

Section: Discussionmentioning

confidence: 99%

“…In Vitro Transcription, Electroporation of HCV Replicons, and Replication Assay-The protocol used for generation and electroporation of HCV RNAs has been described elsewhere (46). For transient replication assays, 400 l of single cell suspensions of (10 7 cells/ml) Huh-7 cells were mixed with 5 g in vitro transcribed subgenomic replicon RNA and transfected by electroporation.…”

Section: Methodsmentioning

confidence: 99%

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A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Dujardin

Madan

Montserret

et al. 2015

Journal of Biological Chemistry

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Evolution of efficacious pangenotypic hepatitis C virus therapies

Ashraf

Iman

Khalid

et al. 2018

Medicinal Research Reviews

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Hepatitis C compromises the quality of life of more than 350 million individuals worldwide. Over the last decade, therapeutic regimens for treating hepatitis C virus (HCV) infections have undergone rapid advancements. Initially, structure-based drug design was used to develop molecules that inhibit viral enzymes. Subsequently, establishment of cell-based replicon systems enabled investigations into various stages of HCV life cycle including its entry, replication, translation, and assembly, as well as role of host proteins. Collectively, these approaches have facilitated identification of important molecules that are deemed essential for HCV life cycle. The expanded set of putative virus and host-encoded targets has brought us one step closer to developing robust strategies for efficacious, pangenotypic, and well-tolerated medicines against HCV. Herein, we provide an overview of the development of various classes of virus and host-directed therapies that are currently in use along with others that are undergoing clinical evaluation.

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Inhibition of HCV Replication by Cyclophilin Antagonists Is Linked to Replication Fitness and Occurs by Inhibition of Membranous Web Formation

Cited by 68 publications

References 47 publications

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Evolution of efficacious pangenotypic hepatitis C virus therapies

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