Structural analysis of herpes simplex virus by optical super-resolution imaging

Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

Cell lines and viruses. HaCaT cells 58 were grown in Glasgow minimal essential medium supplemented with 10% FCS, 2 mM glutamine, 100 U ml À 1 penicillin and 100 g ml À 1 streptomycin. Vero cells (from ATCC) and telomerase immortalized human foreskin fibroblasts (HFF-TERT 59 ) were maintained in DMEM completed with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U ml À 1 penicillin and 100 g ml À 1 streptomycin. A bacterial artificial chromosome cloned HSV-1 genome isolate (strain KOS) was used in these studies and was described previously 60 . VP26-mTurquoise and EYFP-gM viruses were prepared using Red Recombination of bacterial artificial chromosome cloned HSV-1 genome 61 by replacing amino acids 1-4 of VP26 with mTurquoise and adding EYFP(A206K) to the C-terminus of gM. To prepare purified viruses, HaCaT cells were infected at 0.01 plaqueforming unit (PFU) per cell and incubated for 3 days. Supernatants were collected and cleared by low-speed centrifugation. The viruses obtained from the culture supernatants were pelleted at 24,000 g for 2 h and resuspended in PBS with 1% FBS. Viruses were further purified by 5-15% Ficoll gradient ultracentrifugation at 17,500 g for 1.5 h. Visible virus band was collected and spun down at 49,000 g for 2 h. Pellet was resuspended in PBS, aliquoted and stored at À 70°C. Virus titre was assessed by titration in Vero cells.