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

Gondar, Virgínia; Molina‐Jiménez, Francisca; Hishiki, Takayuki; Garcı́a-Buey, Luisa; Koutsoudakis, George; Shimotohno, Kunitada; Benedicto, Ignacio; Majano, Pedro L.

doi:10.1128/jvi.00577-15

Cited by 29 publications

(19 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…One report showed that lack of ApoE in 293T cells completely prevents HCV cell-to-cell transmission, suggesting an essential role (31), whereas another report using Huh7.5.1 cells described that knockdown of ApoE is not involved in this type of HCV transmission because of the observation of successful cell-to-cell transmission, albeit with smaller focus size (32). Recently, another report confirmed that knockdown of ApoE in Huh7.5 cells significantly decreased focus size, and this effect is caused only by ApoE expression depletion in infection donor cells (50), which is in agreement with our finding. For HCV cell-to-cell transmission, ApoE was found to be an important but not an essential factor in Huh7.5 or Huh7.5.1 cells, which is in contrast to the finding derived from 293T cells.…”

Section: Discussionmentioning

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

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

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

View full text Add to dashboard Cite

show abstract

“…As for cell-free entry, SR-BI-independent HCV cell-to-cell transmission has been reported [129]. Studies using apoE-silenced donor cells demonstrated that apoE also plays an important role in this process [130][131][132] while apoE expressed by the recipient cells does not appear to be relevant for HCV cell-to-cell transmission [131,132]. Since apoE is required for a late step in the morphogenesis of viral particles and their infectivity [130,133,134], this suggests that mature enveloped viral particles are transferred between adjacent hepatocytes.…”

Section: Viral and Host Factors Involved In Viral Cell-to-cell Transmmentioning

confidence: 99%

Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process

Colpitts

Tsai

Zeisel

2020

IJMS

View full text Add to dashboard Cite

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver disease worldwide. Its tissue and species tropism are largely defined by the viral entry process that is required for subsequent productive viral infection and establishment of chronic infection. This review provides an overview of the viral and host factors involved in HCV entry into hepatocytes, summarizes our understanding of the molecular mechanisms governing this process and highlights the therapeutic potential of host-targeting entry inhibitors.

show abstract

“…Some studies suggest that VLDL secretion is dispensable for HCV egress and favor a role of ApoE in HCV maturation and egress (22,27,28). It has been suggested that only ApoE is required for the production of trans-complemented HCV particles from nonhepatic cells and for the direct cell-to-cell transmission of HCV (29,30). Small interfering RNA (siRNA) screens identified several components of the secretory pathway, including ER-to-Golgi transport, lipid and protein kinases involved in vesicle budding from the trans-Golgi network (TGN), adaptor proteins, and the recycling endosome, for HCV secretion (31).…”

mentioning

confidence: 99%

Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

Syed

Khan

Yang

et al. 2017

J Virol

View full text Add to dashboard Cite

Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP).In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogoldlabeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route.IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets.

show abstract

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

Cited by 29 publications

References 56 publications

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

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

Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process

Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

Contact Info

Product

Resources

About