FIG 1.

Separation of EVs from HSV-1 virions. (A and B) Schematic of the procedures used for EV isolation. HEL cells were infected with HSV-1(F) (0.1 PFU/cell). At 48 h postinfection, the supernatant was collected, centrifuged at 1,200 rpm for 5 min followed by a centrifugation at 3,500 rpm for 20 min, and filtered through a 0.45-μm filter. The supernatant was concentrated using Centricon Plus 70 (100-kDa cutoff) (Millipore). The sample was then loaded on top of an iodixanol/sucrose gradient ranging from 6 to 18%, with a 1.2% increment in the concentration of iodixanol for a total of 11 different concentrations. The 60% iodixanol was diluted in 10 mM Tris (pH 8) and 0.25 M sucrose. Samples were centrifuged in an SW41Ti rotor for 135 min at 250,000 × g and 4°C in a Beckman Coulter OPTIMA XPN-80 ultracentrifuge. Fractions of 500 μl were collected from the top to the bottom of the gradient. (C) Equal volumes from the fractions were separated in denaturing polyacrylamide gels and analyzed by immunoblot analysis using antibodies against EV components such as CD63, Hrs, Alix, and ARF6, viral tegument proteins such as Us11, VP22, VP16, and ICP0, viral capsid proteins such as UL38, viral envelope proteins such as glycoprotein M (gM) and gD, and a component of the viral replication machinery, UL42. Numbers on the left are molecular weight markers.