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Figure 9 | Virology Journal

Figure 9

From: Intracytoplasmic stable expression of IgG1 antibody targeting NS3 helicase inhibits replication of highly efficient hepatitis C Virus 2a clone

Figure 9

Intracellular antibody expression by adenovirus vector inhibits HCV replication in a sub-genomic GFP based stable replicon cell line. Upper Panel shows flow cytometric analysis of HCV-GFP protein level in replicon cell line expressing intracellular NS3 antibody from adenovirus vector. 1 × 106 replicon cells were seeded in a 100 mm tissue culture dishes and next day they were infected with recombinant adenovirus (Ad-IgG or Ad-Luc). After 72 hours, cells were harvested by using trypsin-EDTA and then analyzed by a flow cytometer where GFP expressing cells were quantified. Lower panel shows that intracellular antibody expression eliminates HCV GFP expression in replicon cell line in a co localization experiment. Replicon cells (2 × 104) were grown in a 2-well chamber slide and after 24 hours cells were infected with increasing concentration of Ad-IgG (ranged from 50, 500, 1000, 2000 and 4000 MOI). After 72 hours, cells were fixed for 15 min with 2% paraformaldehyde. This fixation did not destroy GFP expression. Intracellular expression of antibody was performed using phycoerythrin conjugated anti-human IgG1 antibody (1:50 dilution). After this step, cells were washed and counterstained with hoechst dye. Multiple areas in the slide were randomly examined. Three sets of pictures from the same area were taken. First, the intracellular antibody expression (red), second for the GFP expression and third blue nuclear staining of stained cells under a fluorescent microscope. Finally, the combined picture in the same area was composed using Abode Photoshop (version 7.0) computer software. A: Shows fairly high-level expression of HCV GFP the expression in S-3/GFP replicon cells. B-F: Co-localization of GFP and intracellular antibody. The expression of GFP was decreased in cells infected with Ad-IgG and panel (B-E) the co-localization HCV-GFP (green) and intracellular antibody (red).

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