Once S1R downregulation was verified, cell sellckchem growth was monitored by counting control and S1R-silenced cell suspensions, plating equal cell numbers, and counting the cells again 3 and 5 days later. shRNA constructs that led to differences of more than 25% were discarded. Western blot analysis. Total protein samples were prepared by lysis of the cells in 1�� Laemmli loading buffer. Samples were subjected to SDS-PAGE and transferred into a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with either 5% nonfat milk or 3% bovine serum albumin in PBS-0.25% Tween 20 for 1 h at room temperature and subsequently incubated with primary antibody dilutions in the same buffer for an additional hour (except for goat anti-S1R S-18, which was incubated for 5 h). The membranes were washed for 1 h in PBS-0.
25% Tween 20, incubated with appropriate dilutions of the corresponding secondary antibody, washed for 1 h in PBS-0.25% Tween 20, and developed using enhanced chemiluminescence (ECL). Nonsaturated films were scanned, and relative density values were measured using Image J software (47). Preparation of virus stocks. JFH-1 and D183 viruses were propagated in Huh-7.5.1 clone 2 cells by inoculation at low multiplicity (multiplicity of infection [MOI] = 0.01) (11). Cell supernatants were collected at different times postinoculation (p.i.), and the titers were determined using endpoint dilution and immunofluorescence microscopy as previously described (46). Infection experiments.
For single-cycle infection experiments, Huh-7 cells expressing different lentiviral constructs were plated at a density of 5 �� 104 cells per well in 12-well plates (Corning, Lowell, MA). The next day, the cells were inoculated with D183 virus stocks at an MOI of 10 and incubated at 37��C. Five hours later, the inoculum was removed and the cells were washed twice with warm phosphate-buffered saline (PBS). Twenty-four and 48 h later, samples of the cells and the supernatants were collected for infectious-virus titer analysis (see above) and HCV RNA quantitation (by RT-quantitative PCR [qPCR]). Intra- and extracellular HCV infectivity titers were determined as previously described (46). Persistently infected cell cultures were generated by inoculation of Huh-7 cells with JFH-1 virus (MOI = 0.01).
The cells were cultured until infection was propagated to nearly 100% of the population as revealed by immunofluorescence microscopy, typically at days 12 to 14 postinoculation (36). Influenza virus and vesicular stomatitis virus infections were performed by inoculating Huh-7 cells at an MOI of 10 with Batimastat a virus diluted in PBS with 5 ��g/ml bovine serum albumin (BSA) for 30 min at room temperature. The inoculum was removed, and the cells were replenished with complete growth medium. Protein samples were collected at different time points and analyzed by Western blotting using specific antibodies. Analysis of viral entry using HCVpp.