Figure 3.

Expression of ZIKV capsid protein reduces peroxisome numbers. (A) U251 cells were infected with ZIKV PRVABC59 (MOI = 1). Forty-eight hours later, cell lysates were subjected to immunoprecipitation (IP) with rabbit anti-ZIKV capsid, rabbit anti-PEX19, or rabbit IgG followed by SDS-PAGE and immunoblotting (IB) with antibodies to PEX19 or ZIKV-capsid. WCL, whole-cell lysate. (B) HEK293T cells were transfected with a plasmid encoding FLAG-tagged ZIKV capsid or empty vector (pcDNA3.1) for 48 h. Cell lysates were subjected to SDS-PAGE and immunoblotting with antibodies to PEX19, ZIKV-capsid and actin. The relative levels of PEX19 (compared to actin) from three independent experiments were averaged and plotted. Error bars represent standard error of the mean. * p < 0.05. (C) U251 cells were transfected with a plasmid encoding FLAG-tagged ZIKV capsid or empty vector (pcDNA3.1) for 48 h and then processed for confocal microscopy. Peroxisomes were detected with a rabbit polyclonal antibody to the tri-peptide SKL and donkey anti-rabbit IgG conjugated to Alexa Fluor 546. Transfected cells expressing capsid were detected with a mouse anti-FLAG epitope antibody and donkey anti-mouse IgG conjugated to Alexa Fluor 488. Nuclei were stained using DAPI. Images were obtained using spinning disc confocal microscopy. The relative numbers of peroxisomes (SKL-positive structures) in cells transfected with or without ZIKV capsid plasmid were determined using Volocity image analysis software. Averages were calculated from three independent experiments, in which a minimum of 20 cells for each sample were analyzed. The average number of peroxisomes in mock-treated cells was normalized to 1.0. Bars represent standard error of the mean. ** p < 0.01.