Cell Death-Inducing DFFA-Like Effector b Is Required for Hepatitis C Virus Entry into Hepatocytes

Human TIM and TAM family proteins were recently found to serve as phosphatidylserine (PS) receptors which promote infections by many different viruses, including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection by hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be restored completely by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by PS but not by phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS exposed on the HCV envelope.IMPORTANCE TIM family proteins were recently found to enhance infections by many different viruses, including several members of the Flaviviridae family. However, their importance in HCV infection has not previously been examined experimentally. The TIM family proteins include three members in humans: TIM-1, TIM-3, and TIM-4. The findings derived from our studies demonstrate that TIM-1, but not TIM-3 or TIM-4, promotes HCV infection by functioning as an HCV attachment factor. Knockout of the TIM-1 gene resulted in a remarkable reduction of HCV cell attachment and infection. PS-containing liposomes blocked HCV cell attachment and subsequent HCV infection. HCV particles could also be precipitated with a PS-specific monoclonal antibody. These findings suggest that TIM-1 and its binding ligand, PS, may serve as novel targets for antiviral intervention. KEYWORDS hepatitis C virus, TIM-1, attachment, infection, receptor, TIM-3, TIM-4 H epatitis C virus (HCV) is an enveloped RNA virus containing a 9.6-kb singlestranded RNA genome of positive polarity (1). It is the prototype member of the Hepacivirus genus in the Flaviviridae family (2, 3). The viral RNA genome consists of a long open reading frame (ORF), encoding a single polyprotein, and untranslated regions (UTRs) at both the 5= and 3= ends. Upon translation, the viral polyprotein precursor is cleaved by cellular peptidases and the viral NS2/NS3 metalloprotease and NS3/4A serine protease into 10 individual structural and nonstructural (NS) proteins, designated core (C), envelope proteins 1 and 2 (E1 and E2), p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (4). The structural proteins C, E1, and E2 are essential for the formation of HCV particles (5). The NS3 to NS5B proteins are the minimal set of viral proteins required for HCV RNA replication, although a...

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confidence: 99%

TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection

Wang

Qiao

Hou

et al. 2017

“…In mouse cells, the HCV life cycle is blocked or inefficiently supported at multiple steps, in particular, viral entry and HCV RNA replication (reviewed in reference 5). A surprisingly large number of host factors have been shown to be important in the uptake of HCV into human hepatocytes, including glycosaminoglycans (GAGs) present on heparan sulfate proteoglycans (HSPGs) (6), low-density-lipoprotein receptor (LDLR) (7), CD81 (8), scavenger receptor class B member 1 (SCARB1) (9), the tight junction (TJ) proteins claudin-1 (CLDN1) (10) and occludin (OCLN) (11,12), the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2) (13), the cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) (14), transferring receptor 1 (TfR1) (15), the cell death-inducing DFFA-like effector b (CIDEB) (16), and E-cadherin (17). Of those, differences in the sequences of CD81 and OCLN between the murine and human orthologues can at least in part explain the lower efficiency of HCV uptake by rodent versus human cells.…”

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confidence: 99%

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

Ding

Schaewen

Hrebikova

et al. 2017

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

“…While CIDEA and CIDEC are more widely expressed, CIDEB is mostly expressed in liver cells (27) and induced during hepatic differentiation of stem cells (28,29). Although these proteins can induce cell death when overexpressed (27,30,31), gene knockout (KO) experiments with mice indicate that their function relates mostly to lipid metabolism in vivo (32-34).…”

Section: Our Data Demonstrate That Hcv Infection Of Human Hepatocytesmentioning

confidence: 99%

“…A role for CIDEB in very-low-density lipoprotein (VLDL) lipidation, VLDL transport, and cholesterol metabolism in nonprimate cell culture models has been reported (34)(35)(36). We previously characterized a role for CIDEB in a late step of HCV entry into hepatocytes (29). In this study, we investigated the molecular mechanism and biological consequence of HCV-induced downregulation of CIDEB.…”

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confidence: 99%

Hepatitis C Virus-Induced Degradation of Cell Death-Inducing DFFA-Like Effector B Leads to Hepatic Lipid Dysregulation

Lee

Alsagheir

et al. 2016

Self Cite

Individuals chronically infected with hepatitis C virus (HCV) commonly exhibit hepatic intracellular lipid accumulation, termed steatosis. HCV infection perturbs host lipid metabolism through both cellular and virus-induced mechanisms, with the viral core protein playing an important role in steatosis development. We have recently identified a liver protein, the cell deathinducing DFFA-like effector B (CIDEB), as an HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. In this study, we investigated the biological significance and molecular mechanism of this downregulation. HCV infection in a mouse model downregulated CIDEB in the liver tissue, and knockout of the CIDEB gene in a hepatoma cell line results in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very-low-density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis is further supported by the requirement of the HCV core and its LD localization for CIDEB downregulation, which utilize a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB. IMPORTANCE Our data demonstrate that HCV infection of human hepatocytes in vitro and in vivo results in CIDEB downregulation via a proteolytic cleavage event. Reduction of CIDEB protein levels by HCV or gene editing, in turn, leads to multiple aspects of lipid dysregulation, including LD stabilization. Consequently, CIDEB downregulation may contribute to HCV-induced hepatic steatosis.H epatitis C virus (HCV) is a positive-strand RNA virus and a significant human pathogen. Chronic HCV infection causes liver complications, such as steatosis, cirrhosis, and hepatocellular carcinoma. The arrival of new directly acting antivirals (DAAs) has resulted in markedly improved virologic response in patients with access to these new drugs, but the high cost of the new therapy and the low diagnosis rate of HCV-infected individuals present new challenges for hepatitis C management (1). Furthermore, chronic liver damage can persist even after the infection has been cleared, so HCV pathogenesis remains an area of research highly significant for human health.The HCV life cycle and pathogenesis are intimately linked to host lipid metabolism (2). On one hand, lipids are involved in multiple stages of the infection cycle. HCV virions are assembled on lipid droplets (LDs) (3) and associated with host lipoproteins to form lipoviral particles (LVP) for infection (4). The productive entry of HCV is aided by several molecules involved in lipid uptake (5-7); replication of HCV genome critically depends on a lipid kinase (8, 9) and is regulated by lipid peroxidation (10). On the other hand, HCV infection profoundly disturbs lipid metabolism pathways (11). HCV patients exhibit enhanced lipogenesis (12), consistent with in vitro results showing that HCV infection upregulates genes encoding sterol...