Analysis of fractionated cells expressing wild-type or non-infectious quadruple mutant viruses.

<p>A) Representative buoyant density profile of viral infectivity in cell lysate of stable cell line expressing JFH1-Luc/Neo-wt in continuous 10% to 60% sucrose gradient. Infectivity (black circles) was determined by a focus-forming assay on Huh7.5.1 cells and expressed as log₁₀ of focus-forming units (ffu) per ml of each fraction. Fraction densities were determined by measuring the sucrose content in similar fractions of a control gradient with a refractometer. The dotted line represents the density (g/ml) measured in each fraction. B) Western blotting analysis following sucrose gradient ultracentrifugation of cell lysates from stable cell lines expressing JFH1-Luc/Neo-wt and its quadruple core mutant (R50A/K51A/R59A/R62A). Cytoplasmic lysates were deposited onto the top of a continuous 10% to 60% sucrose gradient and centrifuged at 29,000 rpm for 16 h at 4°C. Samples of 1 ml were collected from the top of the gradient and 10 µl of each fraction was analyzed by Western blotting with antibodies specific to Core, NS5A, envelope glycoproteins E1 and E2, ApoE (apolipoprotein E), TIP47 (tail interacting protein of 47 kDa), CNX (calnexin) and Actin. C) RT-PCR analysis of fractions 6 and 7 from 10%-60% sucrose gradients described above (B). RNA was extracted via acidic phenol-chloroform extraction of the sucrose fractions (200 µl) followed by LiCl precipitation. RNA (300 ng) from each fraction was tested with two pairs of oligonucleotides: one spanning EMCV-IRES and core nucleotide sequence (positions 3475 and 4213 of JFH1-Luc/Neo replicon) and the other spanning NS5A nucleotide sequence (positions 10092 and 10377 of JFH1-Luc/Neo replicon).