Extended Data Fig. 4: Phylogeny of BTN3 domains and antiviral activity of BTN orthologues and paralogues.

a-d, maximum likelihood Haplorrhini BTN3 gene coding sequence phylogenies of separate domains: IgV (a), IgC (b), PRY (c), SPRY (d) under a K2P+G4 substitution model. Trees are rooted at the C. syrichta branch and node confidence values (10,000 bootstrap replicates) are annotated on each node. Tip shapes are coloured by whether each gene exhibits anti-AIV activity (consistent with Fig. 3a). Phylogenies were visualised using FigTree. e–f, A549 cells were transiently transduced with SCRPSY lentiviruses expressing the indicated BTN proteins and challenged with PR8- or Mallard-GFP. Eight hours post-infection, percentage of RFP-positive cells and its subpopulation of GFP-positive cells was measured by flow cytometry. Data are mean +/− SEM of 2 independent experiments which both gave similar results. Detection of these proteins was not possible using commercially available antibodies, due to their genetic divergence compared to human BTN3A1-3. Therefore, tagging of these BTN genes was attempted by introducing a C-terminal FLAG. Despite their protein expression being successfully detected using an anti-FLAG antibody, the addition of FLAG to human BTN3A3 and other BTN genes resulted in the abolishment of their antiviral activity. Cloning of the genes indicated above was conducted in the same way as those constructs shown in Extended Data Fig. 3.