Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells.

Patients and Methods Human Subjects

All protocols involving human tissue were reviewed and exempted by the Rockefeller University Institutional Review Board.

Isolation and Culture of HFLC

Deidentified fetal livers (16-24 weeks gestation) were procured through Advanced Bioscience Resources (ABR; Alameda, CA) or the Human Fetal Tissue Repository of the Albert Einstein College of Medicine (AECOM; New York, NY). Livers received on ice were washed with hepatocyte wash buffer (HWB) consisting of Williams' E Medium (WEM) plus 10 mM HEPES, 50 μg/mL gentamicin, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Tissue was minced then resuspended in 20-40 mL warm digestion buffer consisting of Hanks Balanced Salt Solution plus 40 mM HEPES, 3.26 mM CaCl 2 , 2 U/mL DNase I Grade II (Roche), and 0.2% Collagenase type IV (Sigma). Tissue was digested for 30 minutes at 37°C, then diluted 1:1 with HWB and gently pushed through 70 μm cell-strainers (BD Biosciences). The suspension was centrifuged at 100 g for 3 minutes and the cell pellet containing large hepatocytes was washed twice by resuspension in 50 mL HWB and centrifugation at 100 g for 4 minutes. Hepatocytes were enriched by 1 g sedimentation in 25 mL HWB for 1 hour at room temperature, followed by additional washing. In some experiments hepatocytes were further enriched by centrifugation through lymphocyte separation medium (Cellgro, Manassas, VA) as described. 33 Hepatocyte yields ranged from 0.5 to 4 × 10 7 cells per tissue and cells were generally >80% viable as assessed by Trypan blue exclusion and collagen attachment. Hepatocytes were plated at ≍1 × 10 5 /cm 2 on 24- or 48-well collagen I-coated plates (BD Biosciences) in WEM containing 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, CA), 2 mM L-glutamine (Invitrogen), 1X ITS Plus (BD Biosciences) and antibiotics. After overnight incubation, adherent cells were washed with WEM, then maintained in Hepatocyte Defined Medium (HDM; BD Biosciences) plus L-glutamine and antibiotics. The culture medium was aspirated and replaced every 2 days.

Lentiviral Vectors V proteins and control protein firefly luciferase

(Fluc) were expressed from a hybrid albumin promoter in a bi-cistronic lentiviral vector 34 modified to express the HCV-dependent fluorescence relocalization (HDFR) cassette TagRFP-NLS-IPS. 32 In this cassette the fluorescent reporter TagRFP is fused to both a nuclear localization sequence (NLS) and the transmembrane domain of the mitochondrially tethered adapter protein IPS-1. 32 Following HCV infection of HDFR-expressing cells, cleavage of IPS-1 by the viral NS3-4A protease 2 leads to migration of the fluorescent reporter from mitochondria to the nucleus, enabling visualization of HCV-infected cells. 32 Vector construction and source of protein-coding sequences are detailed in the Supporting Information and Supporting Fig. S1A . Transduction with Lentiviral Pseudoparticles (PP) PP were prepared by cotransfection of 293T cells with lentiviral and packaging plasmids as described. 15 , 32 HFLC were transduced 1-3 days postplating by incubation for 3-6 hours with PP stocks diluted 1:3 in HDM plus 20 mM HEPES and 4 μg/mL polybrene, then washed and fed with HDM. HCVcc Inocula HCVcc inocula used were the Gaussia luciferase reporter virus Jc1FLAG2 (p7-nsGluc2A) 35 (JC1G) and J6JFH Clone 2, 36 which lacks a reporter but replicates to higher titer in Huh-7.5 cells. Virus stocks were prepared by electroporation of in vitro transcribed RNA into Huh-7.5 cells as described 35 and virus was collected in serum-free medium, or medium containing 1.5 or 10% FBS. For some experiments virus stocks were concentrated using Amicon Ultracel-100K filters (Millipore). Infectious titers of HCVcc inocula were determined by titration on Huh-7.5 cells as described 12 and are expressed as 50% tissue culture infectious doses (TCID 50 ). HFLC were infected for 3 to 6 hours with HCVcc diluted in HDM, then washed and fed with HDM.

Detection of HCV RNA Total

RNA was extracted from washed HFLC using RNEasy Kits (Qiagen). HCV RNA was detected by quantitative RT-PCR using the Eragen MultiCode-RTx method (Eragen Biosciences, Madison, WI) and primers directed to the 5′ untranslated region of the HCV genome. Quantitation of HCV RNA copy number was achieved using a synthetic RNA standard (Apath, Brooklyn, NY). Cytokines and Drugs Recombinant human cytokines, IFN-beta (IFN-β), IL-28A, and IL-29, were from Peprotech (Rocky Hill, NJ). The HCV NS5B polymerase inhibitor 2′ C-methyl adenosine (2′CMA) was the gift of D. Olsen and S. Carroll (Merck Research Laboratories, West Point, PA). Immunofluorescence Analysis (IFA), Immunoblotting, and Enzyme-Linked Immunosorbent Assay (ELISA) IFA 9 and immunoblotting 14 were carried out as described. Antibodies and reagents are detailed in the Supporting Information. Human IL-29 was detected using ELISA kits from eBioscience (San Diego, CA). ELISA for human albumin is described in the Supporting Information.

Time-Lapse Live Cell Imaging

HFLC were plated on collagen-coated optical dishes and maintained in HDM. Images were acquired on a Zeiss Axiovert-200 inverted microscope equipped with an environmental chamber.

Visualization of Tag-RFP in transduced

HFLC was achieved by laser excitation and emission as described. 32

Supplementary material Additional Supporting Information may be found in the online version of this article. Supporting Video 1: Firefly luciferase (Fluc) Supporting Video 2: Parainfluenza virus 5 V protein (PIV5 V) or (C). Supporting Video 3: Measles virus V protein (MV V).