Receptor-dependence of fusion-from-without induced by HSV-1. ANG path virions (MOI of 50) were added to receptor-negative CHO cells (A) or CHO cells expressing either nectin-1 (B) or nectin-2 (C) in the presence of 1 mM cycloheximide. Cells were incubated at 37°C for 3 h, and were then fixed with methanol and stained with Giemsa. Magnification, 4×. Approximately 27% of CHO-nectin-2 cells were fused under these conditions. Results with receptor-negative CHO cells (< 1% fusion) were indistinguishable from CHO-nectin-1 cells. (D) Antibodies to nectin-2 block ANG path-mediated fusion-from-without. Anti-nectin-1 polyclonal antibody R154 or anti-nectin-2 polyclonal antibody R143 was added to CHO-nectin-2 cells in 24 well dishes at 4°C for 30 min. HSV-1 ANG path was added to the monolayers for 37°C for 3 h in the presence of a 1:500 dilution of antibody. Cells were fixed, photographed, and quantified for ANG path-induced FFWO. Experiments were repeated at least three times with similar results.