Cells and viruses
African green monkey kidney (Vero) cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in Dulbecco's modification of Eagle minimal medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Chinese hamster ovary cells (CHO-K1, ATCC) were maintained in Ham's F-12 K medium with 10% FBS, 100 U/ml penicillin, and 100 ug/ml streptomycin (Invitrogen, Carlsbad, CA). CHO-HVEM-12 cells (Montgomery et al., 1996) are CHO-K1 derivatives constitutively expressing full-length human HVEM and were kindly provided by Patricia G. Spear (Northwestern University, IL, USA). Both cell lines were grown in Ham's F-12 K medium supplemented with 10% FBS and 400 μg/ml G418 (Invitrogen).
Wild-type and mutant gD β-galactosidase reporter viruses KOS/tk12 and KOS-Rid1/tk12  were kindly provided by Patricia G. Spear and were propagated and titered on Vero cells.
Soluble protein expression constructs
Sequences encoding sHA102 and sHA162 were amplified by PCR on HVEM plasmid pBEC14 (; a kind gift from Patricia Spear) using the following primers: sHA102/sHA162 sense primer, 5'-CCC AAG CTT GCC ACC ATG GAG CCT CCT GGA GAC TGG GGG CC-3'; sHA102 anti-sense primer, 5'-CCC CTC GAG CTA ATG GTG ATG GTG ATG GTG AGC GCG GCA CGC GGC GCA-3'; sHA162 anti-sense primer, 5'-CCC CTC GAG CTA GTG GTG GTG GTG GTG GTG GGA GCT GCT GGT CCC AGC-3'. Primers were designed to provide a Kozak translational initiation sequence at the start of the open reading frame (ORF) and six histidine residues followed by a TAG stop codon at the end. Underlined sequences indicate recognition sites for the restriction enzymes Hind III and Xho I. Following digestion with Hind III plus Xho I, each PCR product was cloned into pcDNA3.1 (+) (Invitrogen), yielding mammalian expression constructs pHA102 and pHA162.
Expression plasmids for sNec1123 and sgD287 were as previously described .
Production and purification of His fusion proteins
Soluble proteins were expressed in 293 T cells and purified using the Probond™ resin purification system (Invitrogen) according to the manufacturer's protocol. Briefly, DNA transfections were performed with LipofectAmine Plus reagent (Invitrogen) and the cells were then cultured in serum-free media. Culture supernatants were harvested at 72 h post-transfection, mixed with Probond resin, and incubated for 1 hr at 4°C. The resin was washed and bound proteins were eluted.
SDS-PAGE and Western blotting
Purified soluble proteins were electrophoresed on a 12% polyacrylamide-sodium dodecyl sulfate (SDS) gel and blotted onto Protran nitrocellulose membrane (Whatman GmbH, Dassel, Germany). The membrane was blocked with a solution of 3% skim milk in PBS/0.1% Tween-20, and incubated for 1 h at room temperature with anti-His antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in blocking buffer. The membrane was washed three times and treated with horseradish peroxide-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO) for 1 h at room temperature. Peroxidase activity was detected using an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).
HSV entry assay
CHO-K1 cells in suspension (6 × 104 cells/well) were preincubated with KOS/tk12 virus in PBS in a total volume of 40 μl for 1 h at 4°C on a rocking device. Increasing concentrations of soluble proteins (0 - 1,000 nM) were added to the virus/cell mixture and incubated for 1 h at 37°C. The cells were collected by centrifugation, washed twice with PBS, resuspended in 40 μl of Ham's F-12 K medium, and plated in a single well of a 96 well plate. After continued incubation at 37°C for 7 h, virus entry was detected by staining with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) or by colorimetric assay (ONPG, o-nitrophenyl-β-D-galactopyranoside), as previously described . Briefly, infected cell monolayers were fixed with 0.2% glutaldehyde (Sigma) for 15 min at room temperature (RT), washed with PBS, and incubated with 0.2 mg/ml X-gal (Sigma);% infection was determined by staining of sequential 10-fold dilutions of infected cells. For the colorimetric assay, infected monolayers were washed with PBS and lysed in 150 μl of 1% NP-40, 1 mM MgCl2, 50 mM β-mercaptoethanol, and 4 mg/ml ONPG (Sigma). Lysates were incubated at 37°C until a yellow color developed, and absorbance was measured at 405 nM.
Infection inhibition by soluble receptors
Different concentrations of soluble receptors (0-2,000 nM) were preincubated with 6 × 104 pfu of KOS/tk12 in a total volume of 40 μl for 1 h at 4°C. CHO-HVEM-12 cells (6 × 104 cells/well) were added and the mixture was incubated for 1 h at 37°C. The cells were then collected, washed twice with PBS, resuspended in 40 μl of Ham's F-12 K medium, and incubated for 7 h at 37°C. Infection was measured by colorimetric ONPG assay, as described above.
Infection inhibition by sgD287
Experiments to determine the sensitivity of sHA102-mediated infection to competition for sHA102 binding with soluble gD were performed essentially as described previously for sNec1123 (Kwon et al., 2006). Briefly, various amounts of sHA102 and sgD287 were incubated with KOS/tk12 in PBS for 1 h at 4°C on rocking device. A total of 5 × 105 CHO-K1 cells (MOI = 3) were added and the mixture was incubated for 1 h at 37°C. The cells were collected, washed, plated with complete medium, and incubated for another 7 h. Infection was measured by ONPG assay.
Increasing amounts of KOS/tk12 were preincubated with 25 μl/ml heparin for 30 min at 4°C, CHO-K1 cells were added, and the incubation was continued for another 30 min. sHA102 to a final concentration of 500 nM was then added and the mixture was incubated for 1 h at 37°C. The cells were washed, plated, incubated with complete medium for 7 h at 37°C, and infection was determined by ONPG assay.
Endocytosis inhibition assays
KOS/tk12 (MOI = 3) was preincubated with CHO-K1 cells for 30 min at 4°C in the presence of 0.3 M sucrose, sHA102 (500 nM final concentration) was added, and the mixture was incubated for 1 h at 37°C. Alternatively, CHO-K1 cells were incubated with different concentrations of ammonium chloride (NH4Cl) or monensin (Sigma) for 30 min at 4°C, virus was added and the incubation continued for another 30 min at 4°C, and sHA102 was added with incubation for 1 h at 37°C. In each situation, the cells were washed, plated, and incubated for 7 h at 37°C in complete medium containing the respective inhibitory agents. Virus entry was measured by ONPG assay.
All results were performed in triplicate and the values are expressed as means ± SD. P values were calculated by using Student's t test, and statistical significance was defined as P ≤ 0.05.