Zinc finger and carboxyl regions of adenovirus E1A 13S CR3 are important for transactivation of the cytomegalovirus major immediate early promoter byadenovirus

Reactivation of latent cytomegalovirus (CMV) is an important cause of disea se in susceptible patients. We previously demonstrated that an adenovirus e arly gene product can transactivate the CMV major immediate early (IE) prom oter in inflammatory cells. This effect was due to the conserved region 3 ( CR3) of the adenovirus E1A 13S gene product. There are two domains in the C R3 region, a zinc finger (aa 147-177) and a carboxyl (aa 180-188) domain. B oth are crucial for transactivation of downstream promoter elements of aden ovirus in E1A 13S, We sought to determine if either or both of these specif ic domains is also necessary for transactivation of the CMV IE promoter by the adenovirus E1A 13S gene product. We cotransfected T-lymphocyte Jurkat c ells and monocyte/macrophage-like THP-1 cells with plasmids expressing wild -type (WT) or CR3 mutant E1A 13S and a CMV IE chloramphenicol acetyltransfe rase (CAT) reporter construct. With extracts of cells coinfected with E1A M IT set to 100%, mutation in the zinc finger domain, the carboxyl domain, or both domains decreased CMV IE CAT activity by greater than or equal to 96% , In contrast, a mutation in the region between the zinc finger and carboxy l domains reduced CMV IE CAT activity by only 24 to 26%, Mixing studies in jurkat cells confirmed the importance of these domains. We also evaluated t he active site of the CMV IE promoter involved in transactivation in THP-1 cells using CMV IE promoter deletions and single promoter element construct s. These studies showed that progressive deletion of the 19-bp CMV IE repea ts containing cyclic AMP response element binding protein/activating transc ription factor (CREB/ATF) sites resulted in progressive loss of activity. T he importance of this element was confirmed using single promoter elements containing CMV IE 16-, 18-, 19-, and 21-bp repeats. Finally, using a 19-bp single promoter element construct and the CR3 mutants we demonstrated that mutations in the zinc finger (C171S) carboxyl region (S185N) or both region s (C171S/S185N) resulted in significant (83, 94, and 85%) loss of activity. We conclude that the zinc finger and carboxyl domains of the CR3 region of EIA 13S are necessary for transactivation of the CMV promoter and that thi s occurs mainly through activation of the 19-bp CREB/ATF site of the promot er.