Recapitulation of the hepatitis C virus life-cycle in engineered murine cell lines

Vogt, Alexander; Scull, Margaret A.; Friling, Tamar; Horwitz, Joshua A.; Donovan, Bridget M.; Dorner, Marcus; Gerold, Gisa; Labitt, Rachael N.; Rice, Charles M.; Ploß, Alexander

doi:10.1016/j.virol.2013.05.036

Cited by 46 publications

(52 citation statements)

References 68 publications

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“…These results refine and extend previous in vitro and in vivo studies highlighting the fact that ablation of host proteins involved in innate immune signaling facilitates HCV replication in mouse cells (7)(8)(9)(10). Notably, mMAVS does potently restrict HCV replication in mouse cells in spite of its susceptibility to cleavage by the CV NS3-4A protease, which was reported recently (31,32). However, it remained undetermined if the efficiency of cleavage paralleled that of hMAVS and if expression of mMAVS poses a greater degree of restriction on HCV replication than its human ortholog.…”

Section: Discussionsupporting

confidence: 89%

“…Of note, in human cells, HCV NS3-4A also cleaves human TRIF, which transmits Toll-like receptor 3 (TLR-3)-dependent signaling for production of IFN (11). Since there is controversy about whether mouse TRIF is susceptible to cleavage by HCV (32,33), it is possible that poor adaptation of HCV to interfere with TRIF-dependent signaling prevents efficient HCV replication in mouse cells. While resolving this question merits further work, it is also possible that lack of human replication cofactors limits replication and accumulation of HCV proteins to establish sufficient interference with innate immunity.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

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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“…Specifically, residues contained in the second extracellular loops of CD81 and OCLN, which have previously been shown to be critical for HCV entry, are not conserved (18,19). We have previously demonstrated that ectopic overexpression of human CD81 and OCLN enables uptake by mouse cell lines of both hepatic and nonhepatic origin (12,20). Both CLDN1 and SCARB1 are also required, but orthologues from other nonhuman species, such as mouse or hamster, have been shown to support HCV entry.…”

mentioning

confidence: 99%

Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo

Ding

Schaewen

Hrebikova

et al. 2017

J Virol

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Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo.IMPORTANCE At least 150 million individuals are chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can result in progressive liver disease and liver cancer. New antiviral treatments can cure HCV in the majority of patients, but a vaccine remains elusive. To gain a better understanding of the processes culminating in liver failure and cancer and to prioritize vaccine candidates more efficiently, smallanimal models are needed. Here, we describe the characterization of a new mouse model in which the parts of two host factors that are essential for HCV uptake, CD81 and occludin (OCLN), which differ between mice and humans, were humanized. We demonstrate that such minimally humanized mice develop normally, express the modified genes at physiological levels, and support HCV uptake. This model is of considerable utility for studying viral entry in the three-dimensional context of the liver and to test approaches aimed at preventing HCV entry.KEYWORDS animal models, hepatitis C virus, viral entry, viral hepatitis H epatitis C virus (HCV) is a positive-sense, single-stranded RNA virus belonging to the Flaviviridae family, genus Hepacivirus (1). HCV progresses to persistent infection in 70 to 80% of those individuals who become acutely infected (2). Chronic carriers are at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) if they remain untreated. Over a few years, very potent directly acting antivirals (DAAs) which can cure

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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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Recapitulation of the hepatitis C virus life-cycle in engineered murine cell lines

Cited by 46 publications

References 68 publications

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

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

Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo

<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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