Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ~170 million individuals infected and current interferon-based treatment having toxic side-effects and marginal efficacy, more effective antivirals are critically needed1. Although HCV protease inhibitors were just FDA approved, analogous to HIV therapy, optimal HCV therapy likely will require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a promising multi-faceted target for antiviral intervention; however, to date FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1-Like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection as silencing or antibody-mediated blocking of NPC1L1 impairs cell-cultured-derived HCV (HCVcc) infection initiation. In addition, the clinically-available FDA-approved NPC1L1 antagonist ezetimibe2,3 potently blocks HCV uptake in vitro via a virion cholesterol-dependent step prior to virion-cell membrane fusion. Importantly, ezetimibe inhibits infection of all major HCV genotypes in vitro, and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor, but also discovered a new antiviral target and potential therapeutic agent.
Hepatitis C virus (HCV) infection is a global health problem, with nearly 2 million new infections occurring every year and up to 85% of these infections becoming chronic infections that pose serious long-term health risks. To effectively reduce the prevalence of HCV infection and associated diseases, it is important to understand the intracellular dynamics of the viral life cycle. Here, we present a detailed mathematical model that represents the full hepatitis C virus life cycle. It is the first full HCV model to be fit to acute intracellular infection data and the first to explore the functions of distinct viral proteins, probing multiple hypotheses of - and-acting mechanisms to provide insights for drug targeting. Model parameters were derived from the literature, experiments, and fitting to experimental intracellular viral RNA, extracellular viral titer, and HCV core and NS3 protein kinetic data from viral inoculation to steady state. Our model predicts higher rates for protein translation and polyprotein cleavage than previous replicon models and demonstrates that the processes of translation and synthesis of viral RNA have the most influence on the levels of the species we tracked in experiments. Overall, our experimental data and the resulting mathematical infection model reveal information about the regulation of core protein during infection, produce specific insights into the roles of the viral core, NS5A, and NS5B proteins, and demonstrate the sensitivities of viral proteins and RNA to distinct reactions within the life cycle. We have designed a model for the full life cycle of hepatitis C virus. Past efforts have largely focused on modeling hepatitis C virus replicon systems, in which transfected subgenomic HCV RNA maintains autonomous replication in the absence of virion production or spread. We started with the general structure of these previous replicon models and expanded it to create a model that incorporates the full virus life cycle as well as additional intracellular mechanistic detail. We compared several different hypotheses that have been proposed for different parts of the life cycle and applied the corresponding model variations to infection data to determine which hypotheses are most consistent with the empirical kinetic data. Because the infection data we have collected for this study are a more physiologically relevant representation of a viral life cycle than data obtained from a replicon system, our model can make more accurate predictions about clinical hepatitis C virus infections.
E2F and retinoblastoma tumor suppressor protein pRB are important regulators of cell proliferation; however, the regulation of these proteins in vivo is not well understood. In Drosophila there are two E2F genes, an activator, de2f1, and a repressor, de2f2. The loss of de2f1 gives rise to the G 1 /S block accompanied by the repression of E2F-dependent transcription. These defects can be suppressed by mutation of de2f2. In this work, we show that the de2f1 mutant phenotype is rescued by the loss of the pre-mRNA splicing factor SR protein B52. Mutations in B52 restore S phase in clones of de2f1 mutant cells and phenocopy the loss of the de2f2 function. B52 acts upstream of de2f2 and plays a specific role in regulation of de2f2 pre-mRNA splicing. In B52-deficient cells, the level of dE2F2 protein is severely reduced and the expression of dE2F2-dependent genes is deregulated. Reexpression of the intronless copy of dE2F2 in B52-deficient cells restores the dE2F2-mediated repression. These results uncover a previously unrecognized role of the splicing factor in maintaining the G 1 /S block in vivo by specific regulation of the dE2F2 repressor function.Many models attribute a central role in cell cycle regulation to a transcriptional factor, E2F, and retinoblastoma tumor suppressor protein (pRB) (for reviews, see references 5, 10, 16, 21, and 48). pRB is a founding member of a family of negative regulators of cell proliferation. The E2F/pRB module translates the activity of cyclin-dependent kinases (cdk) into the cell cycle transcriptional program, thus linking gene expression to the position of the cell within the cell cycle. In mammalian cells, there are eight E2f genes. E2F1 through E2F6 function as heterodimers with DP proteins, while recently discovered E2F7 and E2F8 do not require DP for their activity (31, 32). The E2F/DP heterodimer is referred to as E2F.E2Fs are very loosely classified into two groups according to their function. E2F1, E2F2, and E2F3a are generally considered activators, whereas E2F3b, E2F4, and E2F5 have the properties of repressors. Accordingly, the combined loss of E2f-1, E2f-2, and E2f-3 in primary mouse fibroblasts results in the repression of E2F-dependent transcription and a block in cell proliferation (50). Recent microarray and chromatin immunoprecipitation studies have led to the appreciation that besides regulation of G 1 -to-S progression, E2F/pRB has many other diverse cellular functions, including regulation of G 2 /M progression, apoptosis, DNA repair, DNA recombination, and differentiation (16). There is also considerable debate over the mechanism by which pRB negatively controls cell proliferation and its tumor suppressor properties. Thus, a large number of related E2F and pRB family members in mammalian cells and recent findings that these proteins have a role beyond the G 1 /S control complicate study of the function and regulation of these proteins in vivo.Drosophila melanogaster has a streamlined version of the mammalian cell cycle regulation. This is attested by the funct...
To gain a more complete understanding of hepatitis C virus (HCV) entry, we initially assessed the rate at which HCV initiates productive attachment/infection in vitro and discovered it to be slower than most viruses. Since HCV, including cell culture-derived HCV (HCVcc), exhibits a broad density profile (1.01 – 1.16 g/ml), we hypothesized that the varying densities of the HCVcc particles present in the inoculum may be responsible for this prolonged entry phenotype. To test this hypothesis, we show that during infection, particles of high-density disappeared from the viral inoculum sooner and initiated productive infection faster than virions of low-density. Moreover, we could alter the rate of attachment/infection initiation by increasing or decreasing the density of the cell culture medium. Together, these findings demonstrate that the relationship between the density of HCVcc and the density of the extracellular milieu can significantly impact the rate at which HCVcc productively interacts with target cells in vitro.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.