Generation of fluorescent HCV pseudoparticles to study early viral entry events-involvement of Rab1a in HCV entry

Mapping Intimacies ◽

10.1101/2021.03.11.434898 ◽

2021 ◽

Author(s):

Chayan Bhattacharjee ◽

Aparna Mukhopadhyay

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Confocal Microscopy ◽

Viral Entry ◽

Envelope Glycoprotein ◽

List Type ◽

Enveloped Viruses ◽

Early Entry ◽

Gfp Tag

AbstractUnderstanding the early events in viral biology holds the key to the development of preventives. In this study fluorescent Hepatitis C Virus pseudoparticles have been generated where the envelope glycoprotein has a GFP tag. Using these pseudoparticles entry assays were conducted where the entry of the pseudoparticles was tracked via confocal microscopy. Using this system, fusion of host and viral membranes is predicted to occur within 15 minutes of entry in HCV. Using cells with a knockdown for Rab1a, HCV trafficking was observed to be altered, indicating a role of Rab1a in HCV trafficking. In conclusion, this study reports the generation and use of fluorescent pseudoparticles which may be used to understand the early events of viral entry. This system may be adapted for the study of other enveloped viruses as well.HighlightsFluorescent HCV pseudoparticles have been created to study early entry events.HCV entry tracking via confocal microscopy reveals fusion within 15 minutes.Rab1a is important for HCV trafficking within the cell.

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Conservation of the Conformation and Positive Charges of Hepatitis C Virus E2 Envelope Glycoprotein Hypervariable Region 1 Points to a Role in Cell Attachment

Journal of Virology ◽

10.1128/jvi.75.12.5703-5710.2001 ◽

2001 ◽

Vol 75 (12) ◽

pp. 5703-5710 ◽

Cited By ~ 131

Author(s):

François Penin ◽

Christophe Combet ◽

Georgios Germanidis ◽

Pierre-Olivier Frainais ◽

Gilbert Deléage ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Viral Entry ◽

Envelope Glycoprotein ◽

Cell Attachment ◽

Hypervariable Region ◽

Amino Acid Sequences ◽

Specific Sequence ◽

Amino Acid Region

ABSTRACT Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.

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Sec24C-Dependent Transport of Claudin-1 Regulates Hepatitis C Virus Entry

Journal of Virology ◽

10.1128/jvi.00629-17 ◽

2017 ◽

Vol 91 (18) ◽

Cited By ~ 6

Author(s):

Peiqi Yin ◽

Ye Li ◽

Leiliang Zhang

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Surface ◽

Viral Entry ◽

Vesicular Transport ◽

Transport Pathways ◽

Junction Protein ◽

Dependent Transport ◽

Insight Into

ABSTRACT Claudin-1 is a hepatitis C virus (HCV) coreceptor required for viral entry. Although extensive studies have focused on claudin-1 as an anti-HCV target, little is known about how the level of claudin-1 at the cell surface is regulated by host vesicular transport. Here, we identified an interaction between claudin-1 and Sec24C, a cargo-sorting component of the coat protein complex II (COPII) vesicular transport system. By interacting with Sec24C through its C-terminal YV, claudin-1 is transported from the endoplasmic reticulum (ER) and is eventually targeted to the cell surface. Blocking COPII transport inhibits HCV entry by reducing the level of claudin-1 at the cell surface. These findings provide mechanistic insight into the role of COPII vesicular transport in HCV entry. IMPORTANCE Tight junction protein claudin-1 is one of the cellular receptors for hepatitis C virus, which infects 185 million people globally. Its cellular distribution plays important role in HCV entry; however, it is unclear how the localization of claudin-1 to the cell surface is controlled by host transport pathways. In this paper, we not only identified Sec24C as a key host factor for HCV entry but also uncovered a novel mechanism by which the COPII machinery transports claudin-1 to the cell surface. This mechanism might be extended to other claudins that contain a C-terminal YV or V motif.

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Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

Journal of Virology ◽

10.1128/jvi.74.21.10055-10062.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 10055-10062 ◽

Cited By ~ 156

Author(s):

Sabina Wünschmann ◽

Jheem D. Medh ◽

Donna Klinzmann ◽

Warren N. Schmidt ◽

Jack T. Stapleton

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Confocal Microscopy ◽

Viral Entry ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Protein Synthesis Inhibitor ◽

Low Density ◽

E2 Protein ◽

Intermediate Density

ABSTRACT Hepatitis C virus (HCV) or HCV–low-density lipoprotein (LDL) complexes interact with the LDL receptor (LDLr) and the HCV envelope glycoprotein E2 interacts with CD81 in vitro. However, E2 interactions with LDLr and HCV interactions with CD81 have not been clearly described. Using sucrose gradient-purified low-density particles (1.03 to 1.07 g/cm3), intermediate-density particles (1.12 to 1.18 g/cm3), recombinant E2 protein, or control proteins, we assessed binding to MOLT-4 cells, foreskin fibroblasts, or LDLr-deficient foreskin fibroblasts at 4°C by flow cytometry and confocal microscopy. Viral entry was determined by measuring the coentry of α-sarcin, a protein synthesis inhibitor. We found that low-density HCV particles, but not intermediate-density HCV or controls bound to MOLT-4 cells and fibroblasts expressing the LDLr. Binding correlated with the extent of cellular LDLr expression and was inhibited by LDL but not by soluble CD81. In contrast, E2 binding was independent of LDLr expression and was inhibited by human soluble CD81 but not mouse soluble CD81 or LDL. Based on confocal microscopy, we found that low-density HCV particles and LDL colocalized on the cell surface. The addition of low-density HCV but not intermediate-density HCV particles to MOLT-4 cells allowed coentry of α-sarcin, indicating viral entry. The amount of viral entry also correlated with LDLr expression and was independent of the CD81 expression. Using a solid-phase immunoassay, recombinant E2 protein did not interact with LDL. Our data indicate that E2 binds CD81; however, virus particles utilize LDLr for binding and entry. The specific mechanism by which HCV particles interact with LDL or the LDLr remains unclear.

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