Chaos and bifurcations of a two-dimensional hepatitis C virus model with hepatocyte homeostasis.

In this paper, we delve into the intricate local dynamics at equilibria within a two-dimensional model of hepatitis C virus (HCV) alongside hepatocyte homeostasis. The study investigates the existence of bifurcation sets and conducts a comprehensive bifurcation analysis to elucidate the system's behavior under varying conditions. A significant focus lies on understanding how changes in parameters can lead to bifurcations, which are pivotal points where the qualitative behavior of the system undergoes fundamental transformations. Moreover, the paper introduces and employs hybrid control feedback and Ott-Grebogi-Yorke strategies as tools to manage and mitigate chaos inherent within the HCV model. This chaos arises due to the presence of flip and Neimark-Sacker bifurcations, which can induce erratic behavior in the system. Through the implementation of these control strategies, the study aims to stabilize the system and restore it to a more manageable and predictable state. Furthermore, to validate the theoretical findings and the efficacy of the proposed control strategies, extensive numerical simulations are conducted. These simulations serve as a means of confirming the theoretical predictions and provide insight into the practical implications of the proposed control methodologies. By combining theoretical analysis with computational simulations, the paper offers a comprehensive understanding of the dynamics of the HCV model and provides valuable insights into potential strategies for controlling and managing chaos in such complex biological systems.

Bystander Effects and Profibrotic Interactions in Hepatic Stellate Cells during HIV and HCV Coinfection.

This study aims to explore the influence of coinfection with HCV and HIV on hepatic fibrosis. A coculture system was set up to actively replicate both viruses, incorporating CD4 T lymphocytes (Jurkat), hepatic stellate cells (LX-2), and hepatocytes (Huh7.5). LX-2 cells' susceptibility to HIV infection was assessed through measurements of HIV receptor expression, exposure to cell-free virus, and cell-to-cell contact with HIV-infected Jurkat cells. The study evaluated profibrotic parameters, including programed cell death, ROS imbalance, cytokines (IL-6, TGF-ß, and TNF-α), and extracellular matrix components (collagen, α-SMA, and MMP-9). The impact of HCV infection on LX-2/HIV-Jurkat was examined using soluble factors released from HCV-infected hepatocytes. Despite LX-2 cells being nonsusceptible to direct HIV infection, bystander effects were observed, leading to increased oxidative stress and dysregulated profibrotic cytokine release. Coculture with HIV-infected Jurkat cells intensified hepatic fibrosis, redox imbalance, expression of profibrotic cytokines, and extracellular matrix production. Conversely, HCV-infected Huh7.5 cells exhibited elevated profibrotic gene transcriptions but without measurable effects on the LX-2/HIV-Jurkat coculture. This study highlights how HIV-infected lymphocytes worsen hepatic fibrosis during HCV/HIV coinfection. They increase oxidative stress, profibrotic cytokine levels, and extracellular matrix production in hepatic stellate cells through direct contact and soluble factors. These insights offer valuable potential therapies for coinfected individuals.

PSPC1 Binds to HCV IRES and Prevents Ribosomal Protein S5 Binding, Inhibiting Viral RNA Translation.

Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by an Internal Ribosome Entry Site (IRES) element located in the 5'UTR of viral RNA. Several RNA Binding proteins of the host interact with the HCV IRES and modulate its function. Here, we demonstrate that PSPC1 (Paraspeckle Component 1), an essential paraspeckle component, upon HCV infection is relocalized and interacts with HCV IRES to prevent viral RNA translation. Competition UV-crosslinking experiments showed that PSPC1 interacts explicitly with the SLIV region of the HCV IRES, which is known to play a vital role in ribosomal loading to the HCV IRES via interaction with Ribosomal protein S5 (RPS5). Partial silencing of PSPC1 increased viral RNA translation and, consequently, HCV replication, suggesting a negative regulation by PSPC1. Interestingly, the silencing of PSPC1 protein leads to an increased interaction of RPS5 at the SLIV region, leading to an overall increase in the viral RNA in polysomes. Overall, our results showed how the host counters viral infection by relocalizing nuclear protein to the cytoplasm as a survival strategy.

Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

Development of an effective single-chain variable fragment recognizing a novel epitope in the hepatitis C virus E2 protein that restricts virus entry into hepatocytes.

Previously, we reported a neutralizing monoclonal antibody, A8A11, raised against a novel conserved epitope within the hepatitis C virus (HCV) E2 protein, that could significantly reduce HCV replication. Here, we report the nucleotide sequence of A8A11 and demonstrate the efficacy of a single-chain variable fragment (scFv) protein that mimics the antibody, inhibits the binding of an HCV virus-like particle to hepatocytes, and reduces viral RNA replication in a cell culture system. More importantly, scFv A8A11 was found to effectively restrict the increase of viral RNA levels in the serum of HCV-infected chimeric mice harbouring human hepatocytes. These results suggest a promising approach to neutralizing-antibody-based therapeutic interventions against HCV infection.

A Comparative Study of Human Pluripotent Stem Cell-Derived Macrophages in Modeling Viral Infections.

Macrophages play multiple roles in innate immunity including phagocytosing pathogens, modulating the inflammatory response, presenting antigens, and recruiting other immune cells. Tissue-resident macrophages (TRMs) adapt to the local microenvironment and can exhibit different immune responses upon encountering distinct pathogens. In this study, we generated induced macrophages (iMACs) derived from human pluripotent stem cells (hPSCs) to investigate the interactions between the macrophages and various human pathogens, including the hepatitis C virus (HCV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Streptococcus pneumoniae. iMACs can engulf all three pathogens. A comparison of the RNA-seq data of the iMACs encountering these pathogens revealed that the pathogens activated distinct gene networks related to viral response and inflammation in iMACs. Interestingly, in the presence of both HCV and host cells, iMACs upregulated different sets of genes involved in immune cell migration and chemotaxis. Finally, we constructed an image-based high-content analysis system consisting of iMACs, recombinant GFP-HCV, and hepatic cells to evaluate the effect of a chemical inhibitor on HCV infection. In summary, we developed a human cell-based in vitro model to study the macrophage response to human viral and bacterial infections; the results of the transcriptome analysis indicated that the iMACs were a useful resource for modeling pathogen-macrophage-tissue microenvironment interactions.

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry.

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

Guanylate-binding protein 1 acts as a pro-viral factor for the life cycle of hepatitis C virus.

Viral infections trigger the expression of interferons (IFNs) and interferon stimulated genes (ISGs), which are crucial to modulate an antiviral response. The human guanylate binding protein 1 (GBP1) is an ISG and exhibits antiviral activity against several viruses. In a previous study, GBP1 was described to impair replication of the hepatitis C virus (HCV). However, the impact of GBP1 on the HCV life cycle is still enigmatic. To monitor the expression and subcellular distribution of GBP1 and HCV we performed qPCR, Western blot, CLSM and STED microscopy, virus titration and reporter gene assays. In contrast to previous reports, we observed that HCV induces the expression of GBP1. Further, to induce GBP1 expression, the cells were stimulated with IFNγ. GBP1 modulation was achieved either by overexpression of GBP1-Wt or by siRNA-mediated knockdown. Silencing of GBP1 impaired the release of viral particles and resulted in intracellular HCV core accumulation, while overexpression of GBP1 favored viral replication and release. CLSM and STED analyses revealed a vesicular distribution of GBP1 in the perinuclear region. Here, it colocalizes with HCV core around lipid droplets, where it acts as assembly platform and thereby favors HCV morphogenesis and release. Collectively, our results identify an unprecedented function of GBP1 as a pro-viral factor. As such, it is essential for viral assembly and release acting through tethering factors involved in HCV morphogenesis onto the surface of lipid droplets.

DAA-mediated HCV cure reduces HIV DNA levels in HCV/HIV coinfected people.

IMPORTANCE: Antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can control virus replication and prolong the life of people living with HIV (PLWH). However, the virus remains dormant within immune cells in what is called the HIV reservoir. Furthermore, 2.3 million PLWH are also coinfected with hepatitis C virus (HCV) and are at risk of developing chronic liver disease and cancer. HCV treatment with direct acting antivirals (DAA) can completely cure the infection in more than 95% of treated individuals and improve their long-term health outcomes. In this study, we investigated how HCV treatment and cure affect the HIV reservoir. We demonstrate the beneficial impact of DAA treatment as it reduces the HIV reservoirs in particular in people infected with HCV before HIV. These results support the need for early ART and DAA treatment in HIV/HCV coinfections.

Roles of epidermal growth factor receptor, claudin-1 and occludin in multi-step entry of hepatitis C virus into polarized hepatoma spheroids.

The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a "late receptor" in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process.

The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity.

Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.

Binding of microRNA-122 to the hepatitis C virus 5' untranslated region modifies interactions with poly(C) binding protein 2 and the NS5B viral polymerase.

Hepatitis C virus (HCV) requires two cellular factors, microRNA-122 (miR-122) and poly(C) binding protein 2 (PCBP2), for optimal replication. These host factors compete for binding to the 5' end of the single-stranded RNA genome to regulate the viral replication cycle. To understand how they interact with the RNA, we measured binding affinities of both factors for an RNA probe representing the 5' 45 nucleotides of the HCV genome (HCV45). Isothermal titration calorimetry revealed two, unequal miR-122 binding sites in HCV45, high-affinity (S1) and low-affinity (S2), differing roughly 100-fold in binding affinity. PCBP2 binds a site overlapping S2 with affinity similar to miR-122 binding to S2. PCBP2 circularizes the genome by also binding to the 3' UTR, bridging the 5' and 3' ends of the genome. By competing with PCBP2 for binding at S2, miR-122 disrupts PCBP2-mediated genome circularization. We show that the viral RNA-dependent RNA polymerase, NS5B, also binds to HCV45, and that the binding affinity of NS5B is increased in the presence of miR-122, suggesting miR-122 promotes recruitment of the polymerase. We propose that competition between miR-122 and PCBP2 for HCV45 functions as a translation-to-replication switch, determining whether the RNA genome templates protein synthesis or RNA replication.

The Epigenetic Controller Lysine-Specific Demethylase 1 (LSD1) Regulates the Outcome of Hepatitis C Viral Infection.

Hepatitis C virus (HCV) alters gene expression epigenetically to rearrange the cellular microenvironment in a beneficial way for its life cycle. The host epigenetic changes induced by HCV lead to metabolic dysfunction and malignant transformation. Lysine-specific demethylase 1 (LSD1) is an epigenetic controller of critical cellular functions that are essential for HCV propagation. We investigated the putative role of LSD1 in the establishment of HCV infection using genetic engineering and pharmacological inhibition to alter endogenous LSD1 levels. We demonstrated for the first time that HCV replication was inhibited in LSD1-overexpressing cells, while specific HCV proteins differentially fine-tuned endogenous LSD1 expression levels. Electroporation of the full-length HCV genome and subgenomic replicons in LSD1 overexpression enhanced translation and partially restored HCV replication, suggesting that HCV might be inhibited by LSD1 during the early steps of infection. Conversely, the inhibition of LSD1, followed by HCV infection in vitro, increased viral replication. LSD1 was shown to participate in an intriguing antiviral mechanism, where it activates endolysosomal interferon-induced transmembrane protein 3 (IFITM3) via demethylation, leading endocytosed HCV virions to degradation. Our study proposes that HCV-mediated LSD1 oscillations over countless viral life cycles throughout chronic HCV infection may promote epigenetic changes related to HCV-induced hepatocarcinogenesis.

A Conserved Domain of HCV E2 Glycoprotein Interacts with Human CD81 and Induces Interferon-Gamma Secretion from Peripheral Blood Mononuclear Cells.

BACKGROUND: Hepatitis C virus (HCV) infection is a global health threat to the public, and vaccines against it are not yet available. The HCV envelope glycoprotein E2 is a key target for anti-HCV vaccines. The majority of previous studies have focused on the hypervariable region and the glycosylation sites of the_HCV structural protein. This study aims to investigate a conserved domain of HCV E2 glycoprotein and explore its potential to induce an immune response against HCV. METHODS: HCV E2 conserved domain (encompassing amino acids 505-702) was prepared in Escherichia coli (E. coli). Peripheral blood mononuclear cells (PBMCs) were isolated from patients with HCV or healthy controls. Interferon-gamma (IFN-γ) enzyme-linked immunosorbent spot assay was conducted to examine the HCV E2-specific immune response as reflected by IFN-γ-secreting cells/106 PBMCs. RESULTS: HCV E2 conserved domain was highly conserved among 25 HCV subtypes, and its recombinant soluble production in E. coli was recognized by anti-HCV E2 monoclonal antibodies. This study characterized in vitro direct interaction between bacterially expressed HCV E2 conserved domain and human CD81 (hCD81). Furthermore, the recombinant HCV E2_conserved domain markedly induced the production of IFN-γ by PBMCs from patients with HCV. Its stimulated specific immune response was significantly different from non-specific peptide controls or PBMCs isolated from healthy controls. CONCLUSIONS: HCV E2 conserved domain directly binds hCD81 and activates the production of IFN-γ in the PBMCs of patients with HCV. Therefore, the conserved domain of HCV E2 glycoprotein may be a new candidate for developing an HCV vaccine.

Depression and Cognitive Dysfunction in Patients with Chronic Hepatitis C: Correlation with Viral Replication in the Peripheral Blood Mononuclear Cells and Cytokines in Serum.

Chronic hepatitis C virus (HCV) infection is commonly associated with depression and cognitive dysfunction, the cause of which could be related to the HCV neuroinvasion and/or state of chronic inflammation. Viral sequences and proteins were previously detected in the brain and since blood leukocytes can cross the blood-brain barrier, they could provide viral access to the CNS. Eighty chronic hepatitis C patients were tested for viral replication in PBMCs (detection of the HCV RNA-negative strand) and serum cytokines. Depression was assessed by the Beck Depression Inventory (BDI), neuroticism by the Eysenck Personality Inventory (N/EPO-R), and anxiety by the State-Trait Anxiety Inventory (STAI) while neurocognitive testing included the Wisconsin Card Sorting Test (WCST), Ruff Figural Fluency Test (RFFT), California Verbal Learning Test (CVLT), and Grooved Pegboard Test (GPT). The HCV RNA-negative strand was detected in PBMCs from 24 (30%) patients and these patients had significantly higher BDI scores (median 12.5 [IQR] 6.3-20.5 vs. median 8.00 [IQR] 3-12; p = 0.013). Both depression and anxiety correlated positively with IL-8 while cognitive flexibility, executive function, problem-solving skills, memory, and motor functioning correlated negatively with some proinflammatory cytokines. Our findings suggest that due to chronic HCV infection, the brain function is negatively affected by both viral replication in PBMCs and by the immune activation state.

Role of hepcidin upregulation and proteolytic cleavage of ferroportin 1 in hepatitis C virus-induced iron accumulation.

Hepatitis C virus (HCV) is a pathogen characterized not only by its persistent infection leading to the development of cirrhosis and hepatocellular carcinoma (HCC), but also by metabolic disorders such as lipid and iron dysregulation. Elevated iron load is commonly observed in the livers of patients with chronic hepatitis C, and hepatic iron overload is a highly profibrogenic and carcinogenic factor that increases the risk of HCC. However, the underlying mechanisms of elevated iron accumulation in HCV-infected livers remain to be fully elucidated. Here, we observed iron accumulation in cells and liver tissues under HCV infection and in mice expressing viral proteins from recombinant adenoviruses. We established two molecular mechanisms that contribute to increased iron load in cells caused by HCV infection. One is the transcriptional induction of hepcidin, the key hormone for modulating iron homeostasis. The transcription factor cAMP-responsive element-binding protein hepatocyte specific (CREBH), which was activated by HCV infection, not only directly recognizes the hepcidin promoter but also induces bone morphogenetic protein 6 (BMP6) expression, resulting in an activated BMP-SMAD pathway that enhances hepcidin promoter activity. The other is post-translational regulation of the iron-exporting membrane protein ferroportin 1 (FPN1), which is cleaved between residues Cys284 and Ala285 in the intracytoplasmic loop region of the central portion mediated by HCV NS3-4A serine protease. We propose that host transcriptional activation triggered by endoplasmic reticulum stress and FPN1 cleavage by viral protease work in concert to impair iron efflux, leading to iron accumulation in HCV-infected cells.

Hepatitis C virus non-structural proteins modulate cellular kinases for increased cytoplasmic abundance of host factor HuR and facilitate viral replication.

Host protein HuR translocation from nucleus to cytoplasm following infection is crucial for the life cycle of several RNA viruses including hepatitis C virus (HCV), a major causative agent of hepatocellular carcinoma. HuR assists the assembly of replication-complex on the viral-3'UTR, and its depletion hampers viral replication. Although cytoplasmic HuR is crucial for HCV replication, little is known about how the virus orchestrates the mobilization of HuR into the cytoplasm from the nucleus. We show that two viral proteins, NS3 and NS5A, act co-ordinately to alter the equilibrium of the nucleo-cytoplasmic movement of HuR. NS3 activates protein kinase C (PKC)-δ, which in-turn phosphorylates HuR on S318 residue, triggering its export to the cytoplasm. NS5A inactivates AMP-activated kinase (AMPK) resulting in diminished nuclear import of HuR through blockade of AMPK-mediated phosphorylation and acetylation of importin-α1. Cytoplasmic retention or entry of HuR can be reversed by an AMPK activator or a PKC-δ inhibitor. Our findings suggest that efforts should be made to develop inhibitors of PKC-δ and activators of AMPK, either separately or in combination, to inhibit HCV infection.

Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus.

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.