Generating Pseudotyped HCV Virus Particles for Infection Assays

The following plasmids were provided by Dr. Takaji Wakita at Tokyo Metropolitan Institute of Neuroscience: Genotype 2a HCV genomic RNA clone pJFH1 (APP1025) (38 (link)). pNL4.3.Luc_RΔenv provided by Dr. N.R. Landau (39 (link)), pHCV_H77_E1_E2(AF009606) Dr. Joe Grove (Addgene) (40 ). For single-round infection assay, human embryonic kidney 293T/17 cells (ATCC, CRL-11268) were co-transfected with pNL4.3.Luc_RΔenv, containing firefly luciferase gene at the nef position (1.35 ug) and genotype 1a pHCV_H77_E1_E2(AF009606) (0.6 ug). Transfection was performed in 293T/17 cells using genejuice (Novagen, USA) transfection kit according to manufacturer's protocol. At day 3 or day 4, pseudotyped HCV virus particles were harvested and filtered over 0.45 um nitrocellulose membrane (SartoriusStedim, Gottingen, Germany). Replicative JFH1-AM120-Rluc in vitro transcribed RNA, containing a luciferase reporter gene, was generated according to manufacturer's instructions (Ambion MEGAscript-kit, ThermoFisher, USA) and electroporated into Huh 7.5 cells as previously described (41 (link)). Virus particles were harvested on day 8 and, TCID50s were determined. The TCID50 of HCV ranged from 2 × 10³ to 4 × 10³.

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Nijmeijer B.M., Eder J., Langedijk C.J., Kaptein T.M., Meeussen S., Zimmermann P., Ribeiro C.M, & Geijtenbeek T.B. (2020). Syndecan 4 Upregulation on Activated Langerhans Cells Counteracts Langerin Restriction to Facilitate Hepatitis C Virus Transmission. Frontiers in Immunology, 11, 503.

Publication 2020

nitrocellulose membrane Ambion MEGAscript-kit

293t cells Assay Cells Clone Embryonic Firefly luciferase Gene position Genomic Genotype Human Infection Kidney Luciferase Membrane Nef gene Nitrocellulose Plasmids Replicative Reporter gene Transfection Virus particles

Corresponding Organization : University of Amsterdam

Other organizations : KU Leuven, Inserm, Aix-Marseille Université, Centre National de la Recherche Scientifique, Centre de Recherche en Cancérologie de Marseille

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Variable analysis

independent variables

Genotype 2a HCV genomic RNA clone pJFH1 (APP1025)
PNL4.3.Luc_RΔenv
PHCV_H77_E1_E2(AF009606)

dependent variables

Firefly luciferase gene expression
TCID50 of HCV

control variables

Human embryonic kidney 293T/17 cells (ATCC, CRL-11268)
Huh 7.5 cells

controls

Positive control: Replicative JFH1-AM120-Rluc in vitro transcribed RNA, containing a luciferase reporter gene, electroporated into Huh 7.5 cells

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