Mutants of human adenovirus type 5 (Ad5) lacking early region 4 (E4) display a complex phenotype that includes a delay in the onset of viral DNA replication in low-multiplicity infections. Studies of viral DNA replication in vitro have not revealed a requirement for E4 products in DNA synthesis and, for most E4 mutants, defects in DNA replication are not apparent at high multiplicities of infection. The effects of E4 mutations on DNA replication therefore may reflect a role for E4 in the regulation of replication rather than in the process of DNA synthesis. The E4 mutant H5dl 1014 carries two deletion mutations that together destroy all E4 open reading frames (ORFs) except ORF 4. Immunoprecipitation measurements of the level of the ORF 4 product confirm that H5dl 1014 accumulates the ORF 4 product in somewhat larger amounts than wild-type Ad5, H5dl 1014 is profoundly defective in viral DNA replication at a multiplicity of infection (50 PFU/cell) and time (24 hr after infection) that permit mutants lacking all seven E4 products to accumulate approximately normal amounts of DNA. In contrast, H5dl 1019, a derivative of H5dl 1014 in which the expression of ORF 4 is prevented by a mutation in the ORF 4 ATG initiator codon, produces DNA normally under these conditions. The product of ORF 4 therefore is necessary for the inhibition of viral DNA replication in H5dl 1014-infected cells. H5dl 1014 also inhibits, in trans, the synthesis of viral DNA by other E4 mutants that lack both E4 ORFs 3 and 6. Viruses that possess either of those ORFs are not subject to inhibition, indicating that the ORF 3 and 6 products antagonize the effect of ORF 4. These observations are consistent with a regulatory role for the E4 ORF 3, 4, and 6 products in vital DNA replication in adenovirus-infected cells.
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