Polar encapsidation of adenovirus DNA: cloning and DNA sequence of the left end of adenovirus type 3

The left-end adenovirus type 3 DNA sequence is very similar to those of other subgroup B adenoviruses, especially in the area between the HinfI site (320 base pairs) and the early-region Ia gene. This segment of the genome has been implicated as necessary for the left-end polarity of adenovirus DNA encapsidation. This segment and the sequences flanking it are compared with the corresponding sequences of adenovirus type 5 and adenovirus type 12, and the extent and pattern of intersubgroup homologies are discussed.     

Structure and function of the adenovirus origin of replication

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.     

An adenovirus agnogene.

The nucleotide sequence of a 550 base pairs long segment, located between map positions 21 and 22.5 in the adenovirus type 2 genome has been determined. S1 nuclease mapping and sequence analysis of cDNA copies of adenovirus mRNA demonstrated that the established sequence includes the i-leader which is spliced to the 5'-end of certain adenovirus mRNAs (1). The i-leader which is 440 nucleotides long contains an open translational reading frame which is preceded by an AUG triplet and which terminates in the third segment of the tripartite leader. A polypeptide with the same molecular weight as predicted from the DNA sequence was identified by in vitro translation of mRNA which had been selected by hybridization to DNA fragments, containing sequences from the i-leader. The results thus suggest that the i-leader, unlike other adenovirus leader segments, is used for translation.     

Identification of two distinct regions within the adenovirus minimal origin of replication that are required for adenovirus type 4 DNA replication in vitro.

The adenovirus type 4 origins of replication are located at each end of the linear, protein-linked viral DNA molecule and consist of the terminal 18 bp of the viral genome. The sequence of the first 8 bp of the viral genome varies among different adenovirus serotypes, but the sequence from bp 9 to 18 is conserved in all human serotypes, suggesting that it may be of critical importance to origin function. Using an in vitro system in which purified fractions or crude extracts of adenovirus type 4-infected HeLa cells can support initiation and elongation on linearized plasmid templates containing cloned origin sequences, we examined the effect of single base changes in positions 9 to 18 of the adenovirus origin on DNA replication in vitro. Changes in positions 12 to 16 have little effect, whereas alterations at positions 9, 10, 11, 17, and 18 all reduce the efficiency of initiation of DNA replication by between 50 and 90%. Our results show that the region from bp 9 to 18 contains two sets of bases essential for DNA replication which are separated by 5 bp in which single base changes can be accommodated. The likely role of the region from bp 9 to 18 as containing the recognition sequence for a DNA-protein interaction essential for viral DNA replication is discussed.     

Tn5 mutagenesis of the transforming genes of human adenovirus type 5

We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn 5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCDl, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region la and the N-terminal region of early region 1b are essential for DNA-mediated transformation.