Nuclear factor III (NFIII) is a HeLa sequence-specific DNA-binding protein that stimulates initiation of adenovirus DNA replication in vitro and may be involved in regulation of transcription of several cellular and viral genes. We have studied the interaction between NFIII and the binding site in the adenovirus type 2 (Ad2) origin in detail by methidiumpropyl-EDTA iron(II) and hydroxyl radical footprinting and by alkylation interference experiments. Our results indicate that (i) the core of the recognition sequence is 5'-TATGATAAT-3'; (ii) both major and minor groove base contacts are detected, and all base pairs in the core are involved in binding; (iii) many backbone contacts are observed divided into a large domain coinciding with the core and a small domain; (iv) contact points are not confined to one side of the DNA helix in contrast to the nuclear factor I (NFI)-binding site; (v) the binding site overlaps the NFI-binding site for at least one nucleotide. A number of Ad2 mutants as well as related binding sites in the origins of other adenovirus serotypes were systematically compared for binding with NFIII. The results are in good agreement with the contact point studies and show that at least one AT base pair is commonly required by NFI and NFIII for optimal binding. The strongest

A co-transfection method was used to identify regions of the Marek's disease virus (MDV) genome that inhibit plaque formation when introduced along with total DNA from MDV-infected chicken embryo fibroblasts into secondary chicken embryo fibroblasts. Co-transfections involving MDV plasmids containing the BamHI-D or BamHI-H regions of the genome inhibited plaque formation more than 10-fold. The inhibitory region was localized to a 222-base-pair region that contains a sequence homologous to the consensus origin of replication of alphaherpesviruses. The region also contains several potential transcriptional regulatory elements, including two CCAAT boxes, one Sp1 binding site, and an octamer element. The sequence of this region has been reported previously. Transfection inhibition was also observed for the BamHI-I2 region, although the effect was weaker.