Figure 2.

Agnoprotein facilitates virion release and enhances virus propagation. (A) RPTE cells transfected with BK WT and ΔAgno genomes were incubated over a 6-day time course, and levels of VP1 protein expression determined by indirect immunofluorescence using Incucyte Zoom software (Essen BioScience, Ann Arbor, MI, USA). Levels of VP1 expression are shown relative to the Day 3 BK WT sample. Significance of the changes were analyzed by Student’s t-test and indicated by ** p <0.01; (B) BK virus lacking agnoprotein fails to release virus into the cell culture media. Whole cell lysates and media samples from RPTE cells transfected with BK WT or ΔAgno genomes were analyzed at 48 and 72 h post-transfection for the VP1 capsid protein. GAPDH served as a protein loading control for the whole cell lysates; (C) RPTE cells were infected with BK WT and ΔAgno and cell-associated and media fractions harvested separately. Fluorescence focus assay was then performed to determine the IU/mL⁻¹ of virus in the cells and supernatant; (D) Effect of the anion channel blocker DIDS is independent of agnoprotein. RPTE cells were infected with BK WT or ΔAgno and treated with dimethyl sulphoxide (DMSO) only (control) or 50–100 μM DIDS at 48 h post infection. Media and cell-associated fractions were harvested separately at 72 h post infection. Infectious virus titers were quantified by fluorescence focus assay on naïve RPTE cells and the proportion of total infectivity released into the media for each condition was calculated. Levels of released infectivity are represented as relative to the untreated BK WT samples. The graph corresponds to an average of three experimental repeats. Significance was analyzed by Student’s t-test and is indicated by an asterix * p <0.05, ** p <0.01.