VIRAL CODED PROTEINS INVOLVED IN ADENOVIRUS DNA REPLICATION (TEMPERATURE SENSITIVE MUTANTS)

Abstract

The adenovirus (Ad) in vitro DNA replication system provided a means for biochemical and genetic analysis of the Ad DNA-negative temperature sensitive (ts) mutants H5ts125, H5ts107 and H5ts149. Ad DNA synthesis was assayed in a system requiring Ad DNA covalently linked at each 5' terminus to a 55 kilodalton (K) terminal protein, various fractions of Ad-infected cytoplasm and an extract of uninfected HeLa nuclei. Partially purified cytoplasmic extracts made from H5ts125 and H5ts107, mutants in the Ad DBP, were shown to be required for chain elongation but not necessary for initiation of Ad DNA replication. The Ad DBP enhanced early elongation to the 26th deoxynucleotide by approximately two-to fourfold in this system. Extracts prepared from HeLa cells infected with H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of an isolated 140K Ad DNA polymerase subunit (Ad Pol) which typically copurified in a noncovalent complex with the 80K terminal protein precursor (pTP). The initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures were more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. A replicative activity representing the pTP-Ad Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. These results, together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140K DNA polymerase, suggest that this protein is virally coded. An 838 base pair Ad DNA fragment representing approximately 25% of this open reading frame has been cloned and expressed in E. coli. The plasmid vector used for cloning allowed the production of a 58K fusion protein containing sequences from lac Z, Ad Pol and chloramphenicol acetyl transferase. The fusion protein was used to immunize rabbits in order to obtain an antibody which recognized antigenic domains of the intact 140K Ad Pol. Hyperimmune serum was shown to react with purified Ad Pol by ELISA.