Amplification of Virus Strains

The strain HSV-1/syn 17⁺/CMV-EGFP/UL43 (CMV–cytomegalovirus, EGFP–enhanced green fluorescent protein, UL–unique long), herein designated as wildtype (wt), was generated from the laboratory strain HSV-1 syn 17⁺ (41) via insertion of a GFP expression cassette into the UL43 locus of the HSV-1 genome (BioVex). The GFP cassette is under the control of a CMV promoter and the ablation of UL43 is not obligatory for HSV-1 replication (42, 43). The HSV-1-GFPΔKan-UL41, herein designated as HSV-1 Δvhs, also possesses an EGFP expression cassette inserted into the UL41 gene locus encoding vhs (kindly provided by Martin Messerle, Hannover Medical School, Germany). The UL41 gene encodes for the viral virion host shutoff (vhs) protein which functions as viral endoribonuclease and degrades cellular and viral mRNAs (44, 45). For amplification of HSV-1 wt and HSV-1 Δvhs virus strains, 90% confluent BHK21 cells in 15 T₁₇₅ cell culture flasks were washed once with PBS and HSV-1-infected in infection medium (RPMI 1640 (Lonza, Switzerland), 20 mM HEPES) supplemented with HSV-1 virions at a multiplicity of infection (MOI) of 0.01. After an infection period of 1 h on an orbital shaker at RT, 20 mL DMEM medium [supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL Penicillin, 100 U/mL Streptomycin and 1% non-essential amino acids (100× stock)] were added per cell culture flask and cells were subsequently incubated at 37°C and 5% CO₂. Four days post infection, supernatants containing HSV-1 particles were separated from cell debris via centrifugation at 2,575 × g at 4°C for 10 min. Afterwards, supernatants were transferred into high speed centrifugation tubes and centrifuged at 39,742 × g at 4°C for 2 h. For resuspension of the virus pellet, pellets were overlaid with 150 μL MNT buffer for virus amplification (30 mM MES, 100 mM NaCl, 20 mM Tris) or 150 μL DMEM without phenol red (high glucose; Sigma-Aldrich, Germany) for particle isolation and stored at 4°C overnight. For the preparation of virus stocks, virus suspension was aliquoted into cryo-vials for storage at −80°C. For L-particle isolation, virus suspension was directly loaded onto a Ficoll gradient (see “Isolation of HSV-1-derived particles”). Virus titration was performed as previously described (46).