Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein

Abstract

A genetic system is described which allows the isolation and propagation of adenovirus mutants containing lesions in early region 2A (E2A), the gene encoding the multifunctional adenovirus DNA-binding protein (DBP). A cloned E2A gene was first mutagenized in vitro and then was introduced into the viral genome by in vivo recombination. The E2A mutants were propagated by growth in human cell lines which express an integrated copy of the DBP gene under the control of a dexamethasone-inducible promoter (D. F. Klessig, D. E. Brough, and V. Cleghon, Mol. Cell. Biol. 4:1354-1362, 1984). The protocol was used to construct five adenovirus mutants, Ad5d1801 through Ad5d1805, which contained deletions in E2A. One of the mutants, Ad5d1802, made no detectable DBP and thus represents the first DBP-negative adenovirus mutant, while the four other mutants made truncated DBP-related polypeptides. All five mutants were completely defective for growth and plaque formation on HeLa cell monolayers. Furthermore, the two mutants which were tested, Ad5dl801 and Ad5d1802, did not replicate their DNA in HeLa cells. The mutant Ad5d1804 encoded a truncated DBP-related protein which contained an entire amino-terminal domain derived from the host range mutant Ad5hr4O4, a variant of Ad5 which multiplies efficiently in monkey cells. While results of a previous stuly suggest that the amino-terminal domain of DBP could act independently of the carboxyl-terminal domain to enhance late gene expression in monkey cells, the Ad5d1804 polypeptide failed to relieve the block to late viral protein synthesis in monkey cells. The mutant Ad5d1802 was used to study the role of DBP in the regulation of early adenovirus gene expression in infected HeLa cells. These experiments show that E2A mRNA levels are consistently reduced approximately fivefold in Ad5d1802-infected cells, suggesting either a role for DBP in the expression of its own gene or a cis-acting defect caused by the E2A deletion. DBP does not appear to play a significant role in the regulation of adenovirus early regions IA, IB, 3, or 4 mRNA levels in infected HeLa cell monolayers since wild-type Ad5and Ad5d1802-infected cells showed very little difference in the patterns of expression of these genes.

Granzyme H destroys the function of critical adenoviral proteins required for viral DNA replication and granzyme B inhibition

“…DBP is a multifunctional protein with at least 10 million copies per infected cell, which plays a variety of roles in the viral life cycle; it is essential for DNA replication and it is implicated in early and late gene expression as well as virion assembly (Klessig and Grodzicker, 1979;Nevins and Winkler, 1980;Babich and Nevins, 1981;Nicolas et al, 1983;Rice and Klessig, 1985;de Jong et al, 2003). DBP can be separated into two domains by limited chymotrypsin treatment ( Figure 4A): (i) a highly phosphorylated N-terminal domain that contains two nuclear localization sequences and (ii) a C-terminal domain that is non-phosphorylated, contains two zinc atoms and is sufficient for all DNA replication functions in vitro.…”

“…DBP can be separated into two domains by limited chymotrypsin treatment ( Figure 4A): (i) a highly phosphorylated N-terminal domain that contains two nuclear localization sequences and (ii) a C-terminal domain that is non-phosphorylated, contains two zinc atoms and is sufficient for all DNA replication functions in vitro. In vivo, DBP-deficient Ad5 mutants are not viable, as are deletion mutants of the C-terminal domain or the first 2-40 amino acids in DBP (Rice and Klessig, 1985;Vos et al, 1989). Given the relevance of DBP in the adenovirus life cycle, it is not surprising that cytotoxic cells have developed a strategy to target this molecule.…”

Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis

“…The titer based on ALP expression was generally 10-fold higher than that based on PFU measurements. Virus dl802 was grown in E2A-complementing gmDBP cells as previously described (27). Viruses dl1004 and dl1010 were grown in E4complementing Vero W162 cells (42).…”

The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition.

“…The EIB mutant H5 110 has only E1B 44 kD disrupted (Babiss and Ginsberg, 1984). The E2A mutant H5 802 contains a deletion in the 72-kD single-stranded DBP (Rice and Klessing, 1985). The E3 mutant H5 7001 is deleted in the sequences spanning 78.4 to 86 m.u.…”