DELETION OF THE E4 REGION OF THE GENOME PRODUCES ADENOVIRUS DNA CONCATENERS

Two mutants containing large deletions in the E4 region of the adenovi rus genome H5dl366 (91.9-98.3 map units) and H2dl808 (93.0-97.1 map un its) were used to investigate the role of E4 genes in adenovirus DNA s ynthesis. Infection of KB human epidermoid carcinoma cells with either mutant resulted in production of large concatemers of viral DNA. Only monomer viral genome forms were produced, however, when mutants infec ted W162 cells, a monkey kidney cell line transformed with and express ing the E4 genes. Diffusible E4 gene products, therefore, complement t he E4 mutant phenotype. The viral DNA concatemers produced in dl366- a nd dl808-infected KB cells did not have any specific orientation of mo nomer joining: the junctions consisted of head-to-head, head-to-tail, and tail-to-tail joints. The junctions were covalently linked molecule s, but molecules were not precisely joined, and restriction enzyme map s revealed a heterogeneous size distribution of junction fragments. A series of mutants that disrupted single E4 open reading frames (ORFs) was also studied: none showed phenotypes similar to that of dl366 or d l808. Mutants containing defects in both ORF3 and ORF6, however, manif ested the concatemer phenotype, indicating redundancy in genes prevent ing concatemer formation. These data suggest that the E4 ORFs 3 and 6 express functions critical for regulation of viral DNA replication and that concatemer intermediates may exist during adenovirus DNA synthes is.