Studying HCV Cell Entry with HCV Pseudoparticles (HCVpp)

Abstract

HCV infection leads in 50 to 80% of cases to chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma. Interferons and the nucleoside analog ribavirin form the basis for treatment but are not sufficiently effective and have numerous side effects. Although about 300 million people worldwide are estimated to be infected, the characterization of HCV biology and associated pathologies and development of new therapeutics have been slow. Systems that support HCV replication and particle formation in vitro have emerged only over the last few years, over 15 years after the discovery of the virus. The available infection models have remained limited to chimpanzee (1) and immunodeficient mice carrying engrafted human liver cells (2). HCV pseudoparticles (HCVpp) were the first in vitro infection system to become available for investigation of entry and neutralization of this major human pathogen. HCVpp are formed by incorporation of the full-length hepatitis C virus glycoproteins E1 and E2 onto lenti- or retroviral core particles. HCVpp have been validated by many research groups, closely mimic the functionality of the wild-type virus in terms of cell entry and neutralization, and have even been used to isolate the recent HCV receptor Claudin-1. HCVpp are a useful model system not only because of the functional conservation of the envelope glycoproteins with those of the wild-type virus, but also because the retro- or lentiviral vectors used to form them offer of a number of significant technical advantages.

Mapping a Region of Hepatitis C Virus E2 That Is Responsible for Escape from Neutralizing Antibodies and a Core CD81-Binding Region That Does Not Tolerate Neutralization Escape Mutations

“…The virus-containing extracellular medium was collected 48 h after transfection and separated from cell debris by passing through a 0.45-m filter. For the neutralization assay, the virus-containing medium was incubated with each HMAb at various concentrations, or with phosphate-buffered saline instead of the antibodies as an infectivity control, plus 4 g/ml Polybrene at 37°C for 60 min (4,39).The HCVpp-antibody mixture was transferred to Huh7.5 cells (8 ϫ 10 3 cells/well) preseeded in 96-well plates, and infected cultures were centrifuged at 730 ϫ g for 2 h at room temperature. After incubation at 37°C in the presence of 5% CO 2 for 15 h, the unbound virus was replaced with fresh complete medium, followed by additional incubation for a total of 72 h. For detection of HCVpp entry, 100 l of reconstituted Bright-Glo (Promega, Madison, WI) was added to each well, followed by 2 min of mixing and light detection in a Veritas microplate luminometer (Turner Biosystems, Sunnyvale, CA).…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…A comprehensive protocol for HCVpp production can be found in the review by Bartosch et Cosset [155]. Briefly, HCVpp are generally produced in human embryonic kidney (HEK) 293T cells co-transfected by three expression vectors ( Figure 2).…”

Characterization of hepatitis C virus pseudoparticles by cryo-transmission electron microscopy using functionalized magnetic nanobeads

“…Significant progress has been made possible with the development of HCV pseudoparticles (HCVpps), consisting of unmodified HCV E1-E2 glycoproteins assembled onto retrovirus core particles (Bartosch & Cosset, 2009;Bartosch et al, 2003a;Drummer et al, 2003;Hsu et al, 2003), and with the isolation of an infectious HCV clone able to replicate and produce virus particles in cell culture (HCVcc) (Lindenbach et al, 2005;Wakita et al, 2005;Zhong et al, 2005). HCVpps are a most convenient surrogate model for HCV, as they can be handled in a Biosafety Level 2 laboratory and their extensive characterization has shown that they closely mimic the early steps of HCV entry Helle & Dubuisson, 2008).…”