FIG. 5.

PKA down-regulates EBNA-2- and EBNA-LP-mediated transactivation of LMP1- and BamC-dependent reporters; PKI reverses this effect. (A) BJAB cells were cotransfected with 10 μg of a −512/+40 LMP1 promoter-luciferase reporter plasmid, pSG-EBNA-2, pSG-EBNA-LP, pPKAcsα, and PKI expression plasmid, un-less indicated by a minus sign or a number. Total amounts of input DNA were balanced by addition of vector plasmid DNA. Luciferase activity was measured 48 h later and was normalized to reporter and empty expression vector control experiment results. (B) BJAB cells were cotransfected with a −512/+40 LMP1 promoter-luciferase reporter plasmid, pSG-EBNA-2, pSG-EBNA-LP, and an expression vector for human PKAcsα, CHO PKAcsα, or catalytic inactive CHO PKAcsα. In these experiments, 5 μg of each plasmid was used, unless indicated by a minus sign or a number. Luciferase activity was measured 18 h later instead of 48 h and was normalized to reporter and empty expression vector control. This result is representative of three independent experiments. (C) Similar experiments are presented with 10 μg of a minimal adenovirus E1b promoter-CAT reporter plasmid that has eight upstream copies of the EBNA-2 responsive, −330 to −430 Cp enhancer (39). CAT activity was measured by [¹⁴C]chloramphenicol acetylation. These results are representative of three independent experiments. In all experiments, total input DNA was balanced by addition of vector plasmid DNA.