FIG. 1.

Entry of HSV-1 into cultured CF. (A) Dose-response analysis of HSV-1 entry into CF. Cultured CF (•), along with wild-type CHO-K1 cells (▪), were plated in 96-well plates and inoculated with twofold serial dilutions of β-galactosidase-expressing recombinant HSV-1(KOS) tk12 ranging from 1 to 180 PFU/cell. After 6 h, the cells were permeabilized and incubated with ONPG substrate for quantitation of β-galactosidase activity expressed from the input viral genome. Enzymatic activity was measured by determining OD₄₁₀. Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. The experiment was repeated three times with similar results. (B) Entry of HSV-1 into cultured CF, human LF cells, and CHO-K1 cells expressing either HVEM or 3-OS HS as a gD receptor. The cells were plated in 96-well dishes and then treated with serial dilutions of β-galactosidase-expressing recombinant HSV-1(KOS) tk12 as described for panel A. The values shown represent the amount of reaction product detected spectrophotometrically at a single input dose of 5 PFU/cell. Data represent the mean ± the standard deviation of results in triplicate wells in a representative experiment. The experiment was repeated three times with similar results. (C) Dark-field image of cultured CF infected with GFP-tagged HSV-1. (D) A section of the deconvolved stack showing GFP-tagged capsids near or at the periphery of DAPI-stained blue nuclei. The vertical line to the right indicates the YZ plane of z sections. The horizontal line at the bottom indicates the XZ plane of z sections. Images were collected on a Zeiss Axiovert 100 M microscope equipped with a 63× objective. (E) Three-dimensional rendering of the entire deconvolved stacks. (F) Optical plane of sections presented at 90°. (G) Optical plane of sections presented at 110°. (H) Plot of maximum light intensity due to GFP-tagged HSV-1 observed per stack going from the top to the bottom of a cell.