Detecting Viral Protein Interactions

pCAGGS expression plasmids encoding H3N2 Vic/75 PB1-luc1, PB2, PA, and the indicated ANP32-luc2 construct were transfected into ~100 000 293 T cells at a ratio of 1 : 1 : 1 : 1 (15 ng per well). Control conditions contained pCAGGS-luc1 and untagged PB1, or pCAGGS-luc2 and untagged ANP32A, respectively, with all other components remaining constant. Empty pCAGGS plasmid was used to ensure total transfected DNA was equal across conditions. Twenty-four hours after transfection, cells were lysed in 50 µl Renilla lysis buffer (Promega) for 1 h at room temperature with gentle shaking (Gaussia and Renilla luciferase share the same substrate). Bioluminescence generated by Gaussia luciferase was measured using the Renilla luciferase kit (Promega) on a FLUOstar Omega plate reader (BMG Labtech). Normalized luminescence ratios (NLR) were calculated by dividing the signal from the potential interacting partners by the sum of the two controls, as described [51 (link)].