In vivo identification of sequence elements required for normal function of the adenovirus major late transcriptional control region

A series of adenovirus type 5 variants were constructed to identify the sequence elements which comprise the major late transcriptional control region in the context of the viral chromosome. The variant chromosomes carried a second copy of DNA sequence derived from the region surrounding the major late mRNA cap site. The reiterated segments replaced the normal transcriptional control region of the E1A gene. By monitoring the rate of E1A transcription subsequent to infection with the variants, it was possible to evaluate the capabilities of the substituted major late elements. A segment derived from -55 to +33 (relative to the major late cap site at +1) functioned for early transcription, in the presence of the E1A enhancer domain, but failed to direct enhanced levels of activity late after infection. A segment from -122 to +33 directed both early and enhanced late transcription. The rate of late E1A transcription directed by this element was about 40% of that displayed by the major late control region at its normal position. Inclusion of additional upstream sequences (to -565) did not increase late transcription rates. Thus, the segment between -122 and —52 contains a region required for normal function of the adenovirus major late control region.