Cell entry, efficient RNA replication, and production of infectious hepatitis C virus progeny in mouse liver-derived cells

Frentzen, Anne; Anggakusuma,; Gürlevik, Engin; Hueging, Kathrin; Knocke, Sarah; Ginkel, Corinne; Brown, Richard J. P.; Heim, Markus H.; Dill, Michael T.; Kröger, Andrea; Kalinke, Ulrich; Kaderali, Lars; Kuehnel, F.; Pietschmann, Thomas

doi:10.1002/hep.26626

Cited by 42 publications

(55 citation statements)

References 21 publications

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“…This is highlighted by the necessity of ectopic expression of a number of human proteins and microRNAs (miRNAs) for reconstitution of the viral life cycle in mouse-derived cell lines (4). The cellular proteins that are necessary for HCV IRES-mediated translation and replication have been identified through small interfering RNA (siRNA) knockdown screens and other independent studies.…”

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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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mentioning

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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“…High-molecular-weight (HMW) poly(I·C) was purchased from InvivoGen. HCV subgenomic replicon (HCV-SGR) RNA was generated in-house by in vitro transcription as described previously (9).…”

Section: Methodsmentioning

confidence: 99%

“…Second, HCV replication in murine embryonic fibroblasts (7,8), in mouse liver-derived cells (9), and in mouse livers in vivo (10) is heavily impaired by innate immune signaling, since inactivation of host molecules involved in viral RNA sensing, innate immune signaling, or responsiveness to interferons substantially increases HCV replication. Therefore, ablation of distinct innate immune signaling molecules combined with overexpression of essential human entry factors has emerged as a valid strategy to allow HCV propagation in mouse cells in vitro and in vivo (9,10). However, this environment is only partially immunocompetent, thus limiting utility for immunological studies.…”

mentioning

confidence: 99%

Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

Anggakusuma

Frentzen

Gürlevik

et al. 2015

J Virol

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Hepatitis C virus (HCV) efficiently infects IMPORTANCEIn this study, we found that HCV NS3-4A similarly diminished both human and mouse MAVS-dependent signaling in human and mouse cells. Therefore, it is unlikely that ineffective cleavage of mouse MAVS per se precludes HCV propagation in immunocompetent mouse liver cells. Hence, approaches to reinforce HCV replication in mouse liver cells (e.g., by expression of essential human replication cofactors) should not be thwarted by the poor ability of HCV to counteract MAVS-dependent antiviral signaling. In addition, we show that mouse MAVS induces both type I and type III IFNs, which together control HCV replication. Characterization of type I or type III-dependent interferon-stimulated genes in these cells should help to identify key murine restriction factors that preclude HCV propagation in immunocompetent mouse liver cells. H epatitis C virus (HCV) infection is associated with chronic liver disease, including hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (1). Recent licensing of directly acting antivirals (DAAs) has considerably improved therapeutic options, and novel drug combinations reach cure rates of more than 90% (2). However, natural or treatment-induced virus elimination does not prevent reinfection by HCV. Moreover, many of ca. 160 million infected individuals are not diagnosed, and the vast majority of HCV patients have not been treated (3). Therefore, development of a prophylactic vaccine that efficiently prevents virus transmission is a major challenge for global control of hepatitis C. However, advances in HCV vaccine research are hampered by a lack HCV-permissive, immunocompetent animal models.HCV, a plus-strand RNA virus and member of the family Flaviviridae, has a narrow species tropism. Besides humans, only chimpanzees are naturally susceptible to chronic HCV infection. However, high costs and ethical concerns severely limit utilization of chimpanzees for research purposes. Thus, understanding of the mechanisms that limit HCV replication in other animals, for instance, mice, is an important step to ultimately develop a robust animal model for HCV. Encouragingly, recent advances have highlighted key prerequisites for HCV infection of murine liver cells. First, occludin and CD81 have been recognized to determine HCV species tropism at the level of viral cell entry (4). Importantly, this blockade can be overcome by engineering mice to express human occludin and CD81 (5) or by adaptation of HCV to usage of mouse CD81 (6). Second, HCV replication in murine embryonic fibroblasts (7,8), in mouse liver-derived cells (9), and

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“…HCV RNA copy in sera (copies/mL) A~200 2.78 ± 0.31 × 10 sues other than the liver (7,26). As the apoE protein is an essential factor for the production of HCV particles, only the liver is expected to produce an infectious virus via the apoE protein (2,7,13,26).…”

Section: Table 1 Copy Numbers Of Transgene and Hcv Rna In The Liver Amentioning

confidence: 99%

“…As the apoE protein is an essential factor for the production of HCV particles, only the liver is expected to produce an infectious virus via the apoE protein (2,7,13,26).…”

Section: Table 1 Copy Numbers Of Transgene and Hcv Rna In The Liver Amentioning

confidence: 99%

<b>Pol I-transcribed hepatitis C virus genome RNA replicates, produces an infectious virus and leads to severe hepatic steatosis in transgenic </b><b>mice </b>

et al. 2015

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Patients chronically infected with hepatitis C virus (HCV) are at risk of developing end-stage liver disease and hepatocellular carcinoma. Development of drugs to inhibit hepatocyte damage and a vaccine against HCV is hampered by the lack of a small animal model. We generated mice in which the viral genome RNA was always present in the hepatocytes using a special transgene. Here we show that the HCV genome RNA transcribed by Pol I polymerase can replicate and produce infectious viruses in mice. We obtained a transgenic mouse with 200 copies per haploid which we named the A line mouse. It produced ~ 3 × 10 6 HCV RNA copies/mL serum, which is at the comparable level as patients with chronic HCV infection. This mouse was immunotolerant to HCV and showed hepatic steatosis without any necroinflammation at the age of 6 months or hepatocellular carcinoma at the age of 15 months. Thus, the A line mouse can be used as an animal model for chronic HCV infection. This will enable better study of the abnormalities in metabolism and signal transduction in infected hepatocytes, and development of drugs that cure abnormalities.Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer in the world. Recent advances in directly acting antivirals have improved virological outcomes. However, clarification of the pathogenesis of chronic HCV infection, development of the drug to cure pathological abnormalities and a vaccine to prevent HCV infection, are hampered by the lack of a small animal model for HCV infection. The reason why HCV infects humans but not mice was thought to be that human hepatocytes have specific receptors and factors for HCV that are absent in mice. Such molecules have been identified as CD81, SCARB1, CLDN1 and OCLN (6,(22)(23)(24). Then, it turned out that mouse has each homologue, Cd81, Scarb1, Cldn1 and Ocln, and that CD81 and OCLN must be human origin while Cldn1 and Scarb1, mouse homologues of CLDN1 and SCARB1, can work for HCV entry (5). Previously, one research group had generated a C57BL/6 transgenic mouse expressing the four human factors under an innate immune system-defective background, injected HCV into the tail vein and observed that only 0.4% of whole hepatocytes were infected (4). However, the virus disappeared 3 months after infection, which did not indicate a chronic infection. Recently, the ICR transgenic mouse in which human CD81 and OCLN are expressed under the alpha fetopro-

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Cell entry, efficient RNA replication, and production of infectious hepatitis C virus progeny in mouse liver-derived cells

Cited by 42 publications

References 21 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

Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

<b>Pol I-transcribed hepatitis C virus genome RNA replicates, produces an infectious virus and leads to severe hepatic steatosis in transgenic </b><b>mice </b>

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