Part I. Main protocol: HCV cell-to-cell spread in non-dividing Huh7 cell cultures

Materials and Reagents

1. Huh7 cells (from Dr. Francis Chisari, The Scripps Research Institute, La Jolla, CA) (Zhong et al., 2005)
   
2. JFH-1 HCVcc (generated as previously described and quantified by limiting dilution titer assay) (Yu and Uprichard, 2010; Knipe and Howley, 2007)
   Note: Other infectious HCVcc clones can be used as well.
   
3. Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, catalog number: MT-10-013-CV )
   
4. Fetal bovine serum (FBS) (Hyclone, catalog number: SH30910.03 )
   
5. Penicillin, streptomycin, L-glutamine (Mediatech, catalog number: MT-30-009-CI )
   
6. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D4540 )
   
7. 1x PBS (Mediatech, catalog number: MT- 21-030-CV )
   
8. Hydrogen peroxide (H₂O₂) (Thermo Fisher Scientific, catalog number: H325 )
   
9. AEC detection substrate (BD Biosciences, catalog number: 551015 )
   
10. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    
11. Ezetimibe (Sequoia Research Products, catalog number: SRP04000e ) prepared in DMSO at 20 mM
    
12. Rabbit anti-human CLDN1 polyclonal antibody (Abcam, catalog number: ab63070 )
    
13. Rabbit anti-human NPC1L1 polyclonal antibody (Santa Cruz, catalog number: sc-67236 )
    
14. Human anti-HCV E2 monoclonal antibody MAb AR3A (from Dr. Mansun Law, The Scripps Research Institute, La Jolla, CA) (Law et al., 2008)
    
15. Mouse anti-HCV NS5A 9E10 monoclonal antibody (from Dr. Charles Rice, Rockefeller University, New York, NY) (Lindenbach et al., 2005)
    
16. HRP-conjugated goat anti-human (Thermo Fisher Scientific, catalog number: 31410 ), goat anti-mouse (Thermo Fisher Scientific, catalog number: 31430 ), and goat anti-rabbit (Thermo Fisher Scientific, catalog number: 31460 ).
    
17. Mouse IgG (Santa Cruz, catalog number: sc-2025 ), rabbit IgG (Santa Cruz, catalog number: sc-2027 )
    
18. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
    
19. Triton X-100 (Sigma-Aldrich, catalog number: T-8787 )
    
20. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
    
21. Lipofectamine RNAiMAX transfection reagent (Life Technologies, catalog number: 13778 )
    
22. OptiMEM (Life Technologies, Invitrogen^TM, catalog number: 31985-070 )
    
23. 50 ml centrifuge tubes (Dot Scientific, catalog number: 451-PG )
    
24. Complete DMEM (cDMEM) (see Recipes)
    

Equipment

1. 96-well BioCoat collagen-coated tissue culture plates (Corning, catalog number: 356698 )
   
2. BioCoat collagen-coated T75 cm² flask (Corning, catalog number: 356485 )
   
3. Inverted microscope
   
4. Hemocytometer
   
5. 37 °C, 5% CO₂ cell culture incubator
   
6. Table top centrifuge (e.g. Sorvall, model: T6000D or Eppendorf, model: 5810 R )
   

Procedure