FUNCTIONAL-ANALYSIS OF THE CAAT BOX IN THE MAJOR LATE PROMOTER OF THESUBGROUP-C HUMAN ADENOVIRUSES

Comparisons among sequences predicted to encode the major late promote r (MLP) of adenoviruses from a wide variety of host species show that an inverted CAAT box is among the most highly conserved transcription elements found in the putative MLPs. The high degree of conservation s uggests that the CAAT box plays an important role in the function of t he MLP in vivo, an idea supported by a previous mutational analysis of the core CCAAT sequence. To address the importance of the CAAT box, i n terms both of quantitative levels of transcription and of specificit y, a further set of mutations was created and examined in the context of the viral genome. One mutation, CAAT5, contains individual changes at five positions, four of which correspond to invariant residues in a CAAT box consensus derived either by computer analysis or empirically . The CAAT5 mutation had no discernible phenotype by itself but when c oupled with the previously described USF0 mutation, which disrupts bin ding of the upstream stimulating factor (USF) but is otherwise phenoty pically silent, gave rise to virus with a severe replication deficienc y. Nuclear run-on assays showed that transcription initiation at the m utant MLP was significantly reduced compared with that of the wild typ e or the virus containing CAAT5 alone. Replication of the double mutan t was laffer than that of the previously described USF0::CCCAT virus, suggesting that the additional mutations in the CAAT box had further l owered the binding of transcription factor CP1 (also called CBF, NF-ET ). Replacement of the CAAT box bg an ATF binding site or an OCT1 bindi ng site had no phenotypic effect in an otherwise wild-type background, but replacement in a USF0::CCCAT background led to only partial resto ration of the wild-type phenotype. The failure to restore the function al redundancy normally exhibited by the CAAT box and the proximal upst ream activating element is consistent with the idea that in the adenov irus MLP the CAAT box is preferred over others as the distal transcrip tional element.