VPS52 KO reduces VP26-GFP in cells and the amount of VP8 present on released cell free virions.

A. Confocal imaging of WT, Clone P (Cas9+/+), and three clones of VPS52KO MDBKs, infected with the GFP tagged BoHV-1 virus and stained with DAPI(nucleus), GFP(VP26), Alexa 594(red for VP8) and Alexa 647 (white for cis Golgi marker GM130) at 10hpi. B. Schematic drawing of the experimental setup to quantify the VP8 and GFP proteins. Same cells used in A infected with BoHV1-GFP at MOI = 3 and incubated for 48 hours. A small portion from each tissue culture supernatant was used for plaque assays and qPCR to obtain genome/pfu ratios with the bulk concentrated by ultra-centrifugation. The virus pellets were lysed along with the cell pellets and all lysates were then assayed by Western Blotting using antibodies against VP8, GFP, and beta actin (cell lysates only). Figure drawn with Biorender (https://www.biorender.com/). C. Sample Western Blot results of pelleted viruses from the supernatants. The ratio of VP8 to GFP banding intensity measured by densitometry is shown at the bottom for each sample after normalization to that of the WT (set at 1). Lysates for viruses collected from the WT and Clone P cells were diluted 10 times with Laemilli buffer before 2ul of each sample were loaded to avoid signal saturation. D. Bar chart of VP8 to GFP ratios of concentrated cell free viruses from each cell line measured as in C (***: pE. genome to pfu ratios of cell free viruses calculated as in Fig 7B and 7C (p = 0.06, n = 3). F. Sample Western Blot results of cell lysates collected as in B blotted with antibodies against VP8, GFP and beta actin. VP8 to GFP ratios for all cell lysates were calculated as in C and normalized to that of the WT cells. G,H,I. Bar charts of VP8 to actin, GFP to actin, and VP8 to GFP ratios for each cell lysate normalized to that of WT cells based on repeated experiments (***: p0.05; n = 2).