Isolation of extracellular virus particles.

For mass spectrometry-based studies, released HSV-1 and BoHV-1 virions and L-particles were gradient purified as described previously (69). Briefly, 10 175-cm² flasks of confluent MDBK or HaCaT cells (approximately 6 × 10⁸ cells in total) were infected with BoHV-1 (strain P8-2) or HSV-1 (strain Sc16) at a multiplicity of 0.02. Once cytopathic effect was advanced (3 to 4 days postinfection), the extracellular medium was collected and centrifuged at 3,000 rpm for 30 min at 4°C in a fixed-angle rotor to remove cell debris. Virus particles were then pelleted from the cleared supernatant at 9,000 rpm for 90 min at 4°C. The particle pellet was resuspended in 0.5 ml phosphate-buffered saline (PBS) and carefully layered onto a preformed 11-ml 5% to 15% (wt/vol) Ficoll gradient in a 13.2-ml thin-wall polyallomer ultracentrifuge tube (Beckman Coulter). Gradients were centrifuged at 12,000 rpm for 2 h at 4°C in an SW41 Ti swinging-bucket rotor in a Sorvall Discovery SE ultracentrifuge. Light and heavy bands were harvested by needle puncture through the side of the tube with a 19-gauge hypodermic needle in a volume of <1 ml, diluted in 10 ml PBS, and pelleted at 25,000 rpm for 1 h at 4°C using the same rotor and ultracentrifuge. The pellets were resuspended in a suitable volume of PBS and stored at −80°C.