Microscopy and high-content imaging

ZsG fluorescence in infected cells was imaged directly using an EVOS digital inverted microscope (Thermo-Fisher, Waltham, MA, USA). For immunofluorescence, cells were fixed in 10% formalin for 30 min, permeabilized in PBS containing 0.1% Triton-X100 (v/v) for 10 min, and blocked with PBS 5% BSA (v/v) for 30 min. Primary antibody was rabbit α-SOSV NP (Genscript, Waltham, MA, USA), visualized with an AlexaFluor 488-labeled secondary antibody (Thermo-Fisher, #A11034). Quantification of total rSOSV protein production in Huh7 cells was performed on infected cells labeled as described above, and further stained with Nuc-Blue and Cell-Mask Red (Thermo-Fisher). Images were collected on the Operetta high-content imaging system (PerkinElmer, Waltham, MA, USA) using a 20× objective, with 16 fields per well analyzed using the Harmony software package (PerkinElmer). Total 488 fluorescence per field was determined, averaged for the well, and then normalized to values from control wells containing rSOSV-infected, mock-treated cells.