Roles of Lipoproteins and Apolipoproteins in Particle Formation of Hepatitis C Virus

Hepatitis C virus (HCV) is a major cause of chronic liver disease, infecting approximately 170 million people worldwide. HCV assembly is tightly associated with the lipoprotein pathway. Exchangeable apolipoprotein E (apoE) is incorporated on infectious HCV virions and is important for infectious HCV virion morphogenesis and entry. Moreover, the virion apoE level is positively correlated with its ability to escape E2 antibody neutralization. However, the role of apoE exchange in the HCV life cycle is unclear. In this study, the relationship between apoE expression and cell permissiveness to HCV infection was assessed by infecting apoE knockdown and derived apoE rescue cell lines with HCV. Exchange of apoE between lipoproteins and HCV lipoviral particles (LVPs) was evaluated by immunoprecipitation, infectivity testing, and viral genome quantification. Cell and heparin column binding assays were applied to determine the attachment efficiency of LVPs with different levels of incorporated apoE. The results showed that cell permissiveness for HCV infection was determined by exogenous apoE-associated lipoproteins. Furthermore, apoE exchange did occur between HCV LVPs and lipoproteins, which was important to maintain a high apoE level on LVPs. Lipid-free apoE was capable of enhancing HCV infectivity for apoE knockdown cells but not apoE rescue cells. A higher apoE level on LVPs conferred more efficient LVP attachment to both the cell surface and heparin beads. This study revealed that exogenous apoE-incorporating lipoproteins from uninfected hepatocytes safeguarded the apoE level of LVPs for more efficient attachment during HCV infection. IMPORTANCEIn this study, a neglected but important role of apoE exchange in HCV LVP infectivity after virus assembly and release was identified. The data indicated that apoE expression level in uninfected cells is important for high permissiveness to HCV infection. Secreted apoE-associated lipoprotein specifically enhances infection of HCV LVPs. apoE exchange between HCV LVP and lipoproteins is important to maintain an adequate apoE level on LVPs for their efficient attachment to cell surface. These data defined for the first time an extracellular role of exchangeable apoE in HCV infection and suggested that exchangeable apolipoproteins reach a natural equilibrium between HCV LVPs and lipoprotein particles, which provides a new perspective to the understanding of the heterogeneity of HCV LVPs in composition. M ore than 170 million people worldwide are infected with hepatitis C virus (HCV). Up to 80% of infected individuals are unable to clear the virus, and persistent infections lead to a high risk of developing liver cirrhosis and hepatocellular carcinoma (1). Direct-acting antivirals (DAA) significantly improved treatment efficiency, but an effective vaccine for the control of new HCV infection is still needed due to the limited access to anti-HCV treatment. Moreover, the emergence of DAA-resistant viruses calls for alternative strategies to control viral breakthrough (2-4).A...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection

Yang

Wang

Chi

et al. 2016

“…However, the concept of virion-free infectivity is under debate because cell-free infection by subgenomic RNA-containing exosomes was not successful (28). Infectious HCV virion assembly requires host apolipoproteins (29,30); however, dependence on host lipoproteins for HCV cell-to-cell transmission is controversial. One report stated that the lack of apolipoprotein E (ApoE) expression in a nonhepatic cell line blocked HCV cell-to-cell transmission (31).…”

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confidence: 99%

Visualizing the Essential Role of Complete Virion Assembly Machinery in Efficient Hepatitis C Virus Cell-to-Cell Transmission by a Viral Infection-Activated Split-Intein-Mediated Reporter System

Zhao

Deng

et al. 2017

Hepatitis C virus (HCV) infects 2 to 3% of the world population and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. Many aspects of HCV study, ranging from molecular virology and antiviral drug development to drug resistance profiling, were supported by straightforward assays of HCV replication and infection. Among these assays, the HCV-dependent fluorescence relocalization (HDFR) system allowed live-cell visualization of infection without modifying the viral genome, but this strategy required careful recognition of the fluorescence relocalization pattern for its high fluorescence background in the cytoplasm. In this study, to achieve background-free visualization of HCV infection, a viral infection-activated split-inteinmediated reporter system (VISI) was devised. Uninfected Huh7.5.1-VISI cells show no background signal, while HCV infection specifically illuminates the nuclei of infected Huh7.5.1-VISI cells with either green fluorescent protein (GFP) or mCherry. Combining VISI-GFP and VISI-mCherry systems, we revisited HCV cell-to-cell transmission with clearcut distinction of donor and recipient cells in a live-cell manner. Independently of virion assembly, exosomes have been reported to transfer HCV subgenomic RNA to initiate replication in uninfected cells, which suggested an assembly-free pathway. However, our data demonstrated that HCV structural genes and the p7 gene were essential for not only cell-free infectivity but also cell-to-cell transmission. Additionally, depletion of apolipoprotein E (ApoE) from donor cells but not from recipient cells significantly reduced HCV cell-to-cell transmission efficiency. In summary, we developed a background-free cell-based reporter system for convenient live-cell visualization of HCV infection, and our data indicate that complete HCV virion assembly machinery is essential for both cell-free and cell-to-cell transmission. IMPORTANCE Hepatitis C virus (HCV) infects hepatocytes via two pathways: cell-free infection and cell-to-cell transmission. Structural modules of the HCV genome are required for production of infectious cell-free virions; however, the role of specific genes within the structural module in cell-to-cell transmission is not clearly defined. Our data demonstrate that deletion of core, E1E2, and p7 genes individually results in no HCV cell-to-cell transmission and that ApoE knockdown from donor cells causes less-efficient cell-to-cell transmission. Thus, this work indicates that the complete HCV assembly machinery is required for HCV cell-to-cell transmission. At last, this work presents an optimized viral infection-activated split-intein-mediated reporter system for easy live-cell monitoring of HCV infection.

“…On the other hand, host factors, especially those involved in lipid metabolism, are also important for the correct assembly of HCV particles. Among them, LDs and lipoprotein-associated proteins, such as apolipoprotein B (ApoB), ApoE, and microsomal triglyceride transfer protein, are required for the efficient formation of HCV particles (9,(13)(14)(15)(16)(17)(18). Recently, it was shown that various exchangeable apolipoproteins (ApoA, ApoC, and ApoE) could compensate for one another and redundantly participate in the formation of infectious HCV par-ticles in ApoB and ApoE double-gene-knockout (BE-KO) Huh7 cells (17).…”

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confidence: 99%

Human Cathelicidin Compensates for the Role of Apolipoproteins in Hepatitis C Virus Infectious Particle Formation

Puig-Basagoiti

Fukuhara

Tamura

et al. 2016

Self Cite

Exchangeable apolipoproteins (ApoA, -C, and -E) have been shown to redundantly participate in the formation of infectious hepatitis C virus (HCV) particles during the assembly process, although their precise role in the viral life cycle is not well understood. Recently, it was shown that the exogenous expression of only short sequences containing amphipathic ␣-helices from various apolipoproteins is sufficient to restore the formation of infectious HCV particles in ApoB and ApoE double-gene-knockout Huh7 (BE-KO) cells. In this study, through the expression of a small library of human secretory proteins containing amphipathic ␣-helix structures, we identified the human cathelicidin antimicrobial peptide (CAMP), the only known member of the cathelicidin family of antimicrobial peptides (AMPs) in humans and expressed mainly in bone marrow and leukocytes. We showed that CAMP is able to rescue HCV infectious particle formation in BE-KO cells. In addition, we revealed that the LL-37 domain in CAMP containing amphipathic ␣-helices is crucial for the compensation of infectivity in BE-KO cells, and the expression of CAMP in nonhepatic 293T cells expressing claudin 1 and microRNA miR-122 confers complete propagation of HCV. These results suggest the possibility of extrahepatic propagation of HCV in cells with low-level or no expression of apolipoproteins but expressing secretory proteins containing amphipathic ␣-helices such as CAMP. IMPORTANCEVarious exchangeable apolipoproteins play a pivotal role in the formation of infectious HCV during the assembly of viral particles, and amphipathic ␣-helix motifs in the apolipoproteins have been shown to be a key factor. To the best of our knowledge, we have identified for the first time the human cathelicidin CAMP as a cellular protein that can compensate for the role of apolipoproteins in the life cycle of HCV. We have also identified the domain in CAMP that contains amphipathic ␣-helices crucial for compensation and show that the expression of CAMP in nonhepatic cells expressing claudin 1 and miR-122 confers complete propagation of HCV. We speculate that low levels of HCV propagation might be possible in extrahepatic tissues expressing secretory proteins containing amphipathic ␣-helices without the expression of apolipoproteins. According to recent estimates, more than 160 million people worldwide are chronically infected with hepatitis C virus (HCV) (1), which causes liver-related pathologies that can result in life-threatening diseases such as cirrhosis and hepatocellular carcinoma (2). Chronic HCV infection is also associated with several extrahepatic manifestations, including immunological disorders such as cryoglobulinemia and B-cell non-Hodgkin lymphoma (3). A vaccine is not yet available, but recent advances in antiviral treatment, improving upon what was for many years the standard therapy of pegylated interferon alpha plus ribavirin, now include several direct-acting antivirals (DAAs) that have increased the rate of sustained virological response (SVR) up to 80 ...