Cells and Viruses
Vero and U2OS cells (American Type Culture Collection, Rockville, MD) were propagated in Dulbecco modified Eagle medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA). CHO-nectin-1 (M3A) cells 
(kindly provided by Roselyn Eisenberg and Gary Cohen, University of Pennsylvania) are a hamster cell line derived from the parental CHO-K1 hamster cell line. CHO-nectin-1 cells are stably transformed with the human nectin-1 gene and the Escherichia coli lacZ
gene under the control of the HSV ICP4 promoter 
. The cells were propagated in Ham F-12 nutrient mixture (Invitrogen) supplemented with 10% fetal bovine serum, 150 µg of puromycin (Sigma, St. Louis, MO)/ml, and 250 µg of G418 sulfate (Fisher Scientific, Fair Lawn, NJ)/ml. Cells were subcultured in nonselective medium prior to use in experiments.
Wild type HSV-1 strain KOS was provided by Priscilla Schaffer (Harvard University). HSV-1 KOS mutant d
120, containing a 4.1-kb deletion in both copies of the ICP4 gene 
, and the Vero-derived, complementing cell line E5 
were kindly provided by Neal DeLuca (University of Pittsburgh, PA). Stocks of d
120 virus were propagated on E5 cells. Wild-type Glasgow strain 17 syn+ (17+) 
and its ICP0 deletion mutant derivative dl
were provided by R. Everett, MRC Virology Unit, Glasgow. 17+ and dl
1403 were propagated and titered on U2OS cells.
SDS-PAGE and Western Blot Analysis of Virions
Extracellular HSV-1 virions were prepared by infecting Vero cells grown to 90% confluence with HSV-1 KOS or d
120 at a multiplicity of infection (MOI) of 0.01. After detection of significant cytopathic effect but prior to complete detachment of the cell monolayer, the virus-containing medium was collected and spun at 1,400×g
to remove cell debris. Where indicated, supernatant was layered onto a 5% sucrose cushion, and virions were pelleted by centrifugation for 1 h at 27,000×g
. Samples in Laemmli buffer were separated by SDS polyacrylamide gel (4–20% gradient) electrophoresis. Gels were blotted onto nitrocellulose and probed with 1 µg of mouse monoclonal antibody (MAb)/ml specific for gB, ICP4, VP5 (MAbs H1359; Virusys, Sykesville, MD, HA018, H1A021; Santa Cruz, respectively), ICP0 (MAb 11060; Virusys) or 0.01 µg/ml MAb 1–21 to VP16 (Santa Cruz). Rabbit monoclonal antibody to Rab7 (MAb 9367; Cell Signaling Technology, Danvers, MA) was used at 1
2000. Anti-HSV rabbit polyclonal antibody (HR50; Fitzgerald Industries, Acton, MA) was added at 25 µg/ml. Nitrocellulose membranes were incubated with horseradish peroxidase-conjugated goat secondary antibody (Pierce, Rockford, IL), developed with enhanced chemiluminescence detection reagents (Pierce), and exposed to X-ray film (Kodak).
Limited Proteolysis of HSV Particles
To investigate the location of viral proteins relative to virions, extracellular HSV-1 KOS or d120 virions were treated with various concentrations of Proteinase K (Sigma) for 15 min on ice. To halt proteolysis, warmed Laemmli buffer was added, and reactions were boiled for 10 min. Samples were analyzed by 4–20% SDS-PAGE and Western blotting.
Virus was added to cell monolayers grown on glass coverslips in 24-well culture dishes at an MOI of 5. At 6 hr post-infection, cultures were fixed in ice-cold methanol and blocked with 1% BSA. 1 µg/ml anti-ICP4 MAb H1A021 was added followed by Alexa 488-labeled goat anti-mouse IgG (Invitrogen). Images were captured with an EVOS FL fluorescence microscope (Life Technologies).
Treatments with Proteasome Inhibitor or Lysosomotropic Agents
MG132 (75 µM; Sigma) and monensin (75 µM; Sigma) stock solutions were prepared in dimethyl sulfoxide and ethanol, respectively, and stored at −20°C. 1.5 M stock solution of ammonium chloride (Sigma) was prepared in water immediately prior to use. Confluent CHO-nectin-1 cell monolayers were grown in 96-well dishes. Growth medium was removed from cells and replaced with medium containing agents or medium containing control concentrations of dimethyl sulfoxide or ethanol. Cultures were incubated for 15 to 30 min at 37°C. Virus was added, and cells were incubated in the constant presence of agent for 6 to 7 h. Entry was measured by beta-galactosidase assay.
Beta-galactosidase Reporter Assay for HSV Entry
Following infection, 0.5% Nonidet P-40 (Sigma) cell lysates were prepared. Chlorophenol red-beta-D-galactopyranoside (Roche Diagnostic, Indianapolis, IN) was added, and the beta-galactosidase activity was read at 595 nm with an ELx808 microtiter plate reader (BioTek Instruments, Winooski, VT). Beta-galactosidase activity indicated successful entry. Mean results and standard deviations were calculated for four replicate samples. Each experiment was performed at least three times with similar results.