Temporal regulation of a complex and unconventional promoter by viral products

The DNA polymerase (dnapol) gene of Autographa californica nuclear polyhedrosis virus presents a complex promoter organization. It lacks the usual TATA box and start site, and its RNA accumulation initially increases and then decreases dramatically during infection. We investigated dnapol temporal regulation. Transiently expressed dnapol gene was transcribed at a low level from minor start sites. Coexpression with ie0 and/or ie1 immediate-early genes dramatically enhanced dnapol transcription, specifically from a new start site. Moreover, the ie1 transactivation required little or no information in front of this nonconventional proximal promoter. We showed that IE0 and IE1 proteins were stably expressed during infection and that the dnapol mRNA level decrease was not a consequence of the disappearance of these proteins. The dnapol promoter region contains a putative overlapping open reading frame (ORF) in the opposite direction. We showed that ORF-2 was indeed highly expressed late, when the dnapol mRNA level decreased, and that during that time, dnapol mRNA stability was not significantly altered, excluding a destabilizing antisense effect. Additionally, we showed that the dnapol promoter was inhibited late but not early during the infection of cells transiently expressing constructs carrying either the intact or the altered ORF-2 promoter. Therefore, ORF-2 initiation of transcription and dnapol promoter inhibition are two coincidental nonrelated phenomena. Finally, we showed that both IE1 transactivation and late inhibition occurred in the same limited region around the dnapol promoter.