Nectin 1

In order to determine the expression profile of HSV receptors on cell surface, human cancer cell lines Hela, A375, A375-HVEM, Mel526 and EC9706 were separately stained with 5 μl of PE conjugated anti--human HVEM, nectin-1 and nectin-2 antibodies (BioLegend, San Diego, CA) in 2% FBS-PBS at 4°C for 30 min. The stained cells in 2% FBS-PBS were analyzed by flow cytometry (BD Biosciences, San Jose, CA).

Cells were dissociated using HyQtase, then stained with primary antibody for 30 min at 4 °C. Following washing, they were incubated with secondary antibody, again for 30 min at 4 °C. Cells were washed, fixed in 4% paraformaldehyde, and data collected on an Attune cytometer (Thermofisher). In one experiment (Figure 1A), cells were fixed and permeabilised (Cytofix/Cytoperm, BD) before staining. Antibodies used were against HLA-ABC (W632; AbD Serotec), NCR3LG1/B7-H6 (MAB7144, Biotechne R&D Systems), Nectin 1 (R1.302; Biolegend), MICA (AMO1-100; BAMOMAB), MICB (BMO2-100; BAMOMAB), ULBP2 (BUMO1; BAMOMAB), Spike (1A9; Insight), Nucleocapsid (1C7; Stratech), anti-mouse IgG AF647 (Thermofisher).

Antibodies for gD (LP14) VP16 (LP1) and glycoprotein M (gM) were kindly provided by Colin Crump. Other antibodies were purchased commercially: nectin1 and nectin2 (Biolegend); α-tubulin (Sigma); GFP (Clontech); ICP0, ICP27 and CD71 (Santa Cruz); and Cbl (Abcam). IRDye secondary antibodies were from LICOR Biosciences.