Fig. 1

Expression of zebrafish (ZF) 3-O-sulfotransferase-3 (3-OST-3) isoform in wild-type Chinese hamster ovary (CHO-K1) cells results in herpes simplex virus type 1 (HSV- 1) entry. (A) Dose–response curve of HSV-1 entry into CHO-K1 cells expressing ZF 3-OST-3 isoform. Resistant wild-type CHO-K1 cells were transfected with ZF 3-OST-3 at 2.5 μg DNA, which in resulted HSV-1 KOS (gL86) entry, similar to human 3-OST-3 (H 3-OST-3) expression. Cells transfected with empty vector pDream2.1 at 2.5 μg DNA was used as a negative control. The enzymatic activity was measured at an optical density of 410nm (OD₄₁₀). Each value shown is the mean of three or more determinations (±standard deviation). (B) HSV-1 entry into ZF 3-OST-3 isoform expressing CHO-K1 cells was confirmed by X-gal staining. CHO-K1 cells (4 x 10⁶ cells) expressing H 3-OST-3 (panel a) and ZF 3-OST-3 (panel b) were challenged with β-galactosidase-expressing recombinant HSV-1 KOS (gL86) at 25 pfu/cell. Wild-type CHO-K1 cells expressing pDream2.1 (panel c) were also infected in parallel as a negative control. After 6 h of infection at 37°C, cells were washed with phosphate-buffered saline, fixed, permeabilized, and incubated with X-gal (5 bromo-4 chloro-3-indoyl-β-D-galactosidase) at 1.0mg/mL, which yields an insoluble blue product upon hydrolysis by β-galactosidase. Blue cells (representing viral entry) were identified. Microscopy was performed using a 20x objective of Nikon D-Eclipse-C1. (C) Observation of HSV-1 entry in cultured CHO-K1 expressing ZF 3-OST-3 isoform. Confluent monolayers of ZF 3OST-3 CHO-K1 cells (panel b) were infected with green fluorescent protein–tagged capsid of HSV- 1 (VP26-green fluorescent protein) at 25 pfu/cell for 90 min. In parallel CHO-K1 cells expressing pDream2.1 (panel c) and H 3-OST-3 (panel a) were used as negative and positive controls, respectively. At 90 min postinfection, cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde, and stained with 10nm rhodamine-conjugated phalloidin (red) for F-actin. The images were taken using a fluorescent confocal microscope (Nikon D-Eclipse-C1) at 40x objective.