FIG. 3.

Involvement of c-fos in expression of BZLF1 mRNA after EBV infection. (A) Effect of c-fos on gene expression from BZLF1 promoter. The BZLF1-luciferase reporter plasmid (pGVB2/BZLF1) and a c-fos-expressing plasmid or control plasmid were cotransfected into EBV-negative Daudi cells by lipofection. After 48 h of incubation, cells were harvested and luciferase (luc) activity was determined. The luciferase activities obtained from c-fos-transfected cells were divided by those from control plasmid-transfected cells, and the results were expressed as the mean relative activity (fold induction) ± standard error. (B) Expression of c-fos in c-fos-silenced EBV-negative Daudi cell clones. EBV-negative Daudi cells were transfected with the TransSilent Fos shRNA vector (Panomics), and c-fos-silenced cell clones were isolated by cultivation in a selective medium. Expression of the c-Fos protein in these cell clones was examined by immunoblot analysis using a rabbit polyclonal antibody (Calbiochem). The β-actin protein was detected to show the quantity of each protein preparation. (C) Effect of c-fos silencing on expression of BZLF1 mRNA and protein after EBV infection. c-fos-silenced, EBV-negative Daudi cells were infected with EBV. At the designated time of incubation, expression of BZLF1 mRNA and protein was examined by RT-PCR and immunoblot analysis. EBV-negative Daudi cell clones stably transfected with the TransSilent control vector (Panomics) were also infected with EBV and examined for BZLF1 mRNA and protein expression as a positive control. Effect of c-fos silencing on EBNA2 mRNA expression was examined by RT-PCR.