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Fig. 9. NuMA knockdown using siRNA results in an HSV growth defect. (A) siNuMA-, siCont- or mock-transfected HEp-2 cells were analysed 3 days post-transfection for protein expression. NuMA levels were greatly decreased in siNuMA-transfected cells, whereas levels of lamin B and actin were constant. (B-G) Analysis of viral cell-to-cell growth in siRNA-treated cells. HEp-2 cells transfected with siCont (B,C,D) or siNuMA (E,F,G) were infected with 14R-VP16G at a MOI of 0.001 PFU/cell, fixed at 48 hours post infection, and examined for VP16-GFP (B,E). DIC (C,F) and merged images (D,G) show that cell-to-cell viral spread was considerably affected in siNuMA-transfected cells and that this was not a consequence of low cell density. (H) Comparison of 14R-VP16G plaque diameters in siCont- and siNuMA-transfected cells. Cells were fixed at 48 hours post infection and 30 randomly chosen plaques were scanned with the confocal microscope followed by measurement of plaque diameter with the Zeiss LSM software. Error bars represent standard deviations. (I) A multi-step growth curve of HSV-1 in siRNA-treated HEp-2 cells. The results shown are representative of three independent experiments. After similar treatment with siRNA, HEp-2 cells were infected with HSV-1 at a MOI of 0.01 PFU/cell. Cells were harvested at various times post infection to obtain viral growth curves and the resulting viral yield was normalised to per 10⁶ cell. Cells that were not transfected with siRNA showed similar growth kinetics as siCont-transfected cells (not shown). The viral growth kinetics were considerably hampered in NuMA knockdown cells.

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