Infection Experiments of Dendritic Cells

Infection experiments of iDCs or mDCs were performed as recently described [82 (link),83 (link)]. Briefly, iDCs or mDCs were harvested and washed in RPMI 1640. Cells (1 × 10⁶–2 × 10⁶) were resuspended in 300–350 µL infection medium (RPMI 1640 supplemented with 20 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; Lonza)) containing a defined amount of HSV-1 or HSV-2 plaque forming units (pfu). For HSV-1 infection, a multiplicity of infection (MOI) of 1 or 2 was used (as indicated), whereas HSV-2 infection experiments were performed using an MOI of 5. As a mock control, cells were treated with infection medium supplemented with the respective amount of MNT buffer (30 mM MES, 100 mM NaCl, and 20 mM Tris). After 1 h of infection at 37 °C and shaking at 300 rpm cells were collected via centrifugation. Subsequently, cells were cultured in DC medium supplemented with 40 U/mL GM-CSF and 250 U/mL IL-4. To block proteasomal degradation either 10 µM MG-132 (Enzo Life Science, Lörrach, Germany) or 2 µM bortezomib (BZ; Santa Cruz Biotechnology, Heidelberg, Germany) were added 1 hpi (hour post infection). For blocking the ubiquitin E1-activating enzyme, 80 µM PYR-41 (Sigma-Aldrich) was added 4 hpi. As solvent control, dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to the medium.