Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

Barretto, Naina; Sáinz, Bruno; Hussain, Snawar; Uprichard, Susan L.

doi:10.1128/jvi.03241-13

Cited by 43 publications

(58 citation statements)

References 63 publications

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“…This is consistent with the report from Barretto et al 33 Engineering nonhepatic cells to produce HCV through ectopic apoE expression can confer cell-to-cell transmission capacity, albeit to a limited degree relative to the levels observed in the current study. 34 We thus cannot exclude that epitopes not recognized by the utilized anti-apoE antibody play a role in cell-to-cell transmission or that this antibody may have limited access to the virus during this process.…”

Section: Discussionsupporting

confidence: 82%

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

et al. 2016

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BACKGROUND & AIMS:Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. METHODS: We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell cultureÀderived HCVÀproducing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell cultureÀderived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein 2 and HCV virions by immunoprecipitation. RESULTS: Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein 2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. CONCLUSIONS: In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines.

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Section: Discussionsupporting

confidence: 82%

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

et al. 2016

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“…However, our results indicate that significant differences exist between the abilities of ApoB and ApoE to mediate this route of infection. It is important to note that a previous study reported that ApoB or ApoE depletion had no effect on HCV cell-cell spread (34). We interpret this divergence in terms of differences in the experimental approaches: e.g., the duration of the experiment (5 days p.i.…”

Section: Discussionmentioning

confidence: 79%

“…However, this is controversial since another study described that ApoE, ApoB, and microsomal triglyceride transfer protein (MTP) are not involved in this type of infection (34). By blocking cell-free infectivity, we show that blocking ApoE in donor cells inhibits cell-to-cell HCV infection.…”

mentioning

confidence: 80%

Apolipoprotein E, but Not Apolipoprotein B, Is Essential for Efficient Cell-to-Cell Transmission of Hepatitis C Virus

Gondar

Molina‐Jiménez

Hishiki

et al. 2015

J Virol

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Hepatitis C virus (HCV) infects hepatocytes through two different routes: (i) cell-free particle diffusion followed by engagement with specific cellular receptors and (ii) cell-to-cell direct transmission mediated by mechanisms not well defined yet. HCV exits host cells in association with very-low-density lipoprotein (VLDL) components. VLDL particles contain apolipoproteins B (ApoB) and E (ApoE), which are required for viral assembly and/or infectivity. Based on these precedents, we decided to study whether these VLDL components participate in HCV cell-to-cell transmission in vitro. We observed that cell-to-cell viral spread was compromised after ApoE interference in donor but not in acceptor cells. In contrast, ApoB knockdown in either donor or acceptor cells did not impair cell-to-cell viral transmission. Interestingly, ApoB participated in the assembly of cell-free infective virions, suggesting a differential regulation of cell-to-cell and cell-free HCV infection. This study identifies host-specific factors involved in these distinct routes of infection that may unveil new therapeutic targets and advance our understanding of HCV pathogenesis. IMPORTANCEThis work demonstrates that cell-to-cell transmission of HCV depends on ApoE but not ApoB. The data also indicate that ApoB is required for the assembly of cell-free infective particles, strongly suggesting the existence of mechanisms involving VLDL components that differentially regulate cell-free and cell-to-cell HCV transmission. These data clarify some of the questions regarding the role of VLDL in HCV pathogenesis and the transmission of the virus cell to cell as a possible mechanism of immune evasion and open the door to therapeutic intervention.

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“…One report stated that the lack of apolipoprotein E (ApoE) expression in a nonhepatic cell line blocked HCV cell-to-cell transmission (31). In contrast, knockdown of ApoE, ApoB, and microsomal triglyceride transfer protein (MTP) did not block efficient cell-to-cell transmission (32). Whether HCV assembly parts play an important role in cell-to-cell transmission has not been clearly determined due to the lack of a straightforward cell system which enables live-cell distinctions between donor and recipient cells during HCV cell-to-cell transmission.…”

mentioning

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

J Virol

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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.

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Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

Cited by 43 publications

References 63 publications

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies

Apolipoprotein E, but Not Apolipoprotein B, Is Essential for Efficient Cell-to-Cell Transmission of Hepatitis C Virus

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

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