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A series of adenovirus type 5 precursor terminal protein (pTP) and DNA polymerase (Ad pol) genes with linker insertion mutations were separately introduced into the vaccinia virus genome under the control of a late genes in vitro and reintroduction of the mutated genes into the Ad genome (17,41). In our studies, a 12-bp synthetic oligonucleotide was inserted into multiple restriction sites 265 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from supernatant was used as virus stock for all further experiments. Titers of wt and mutant virus stocks were measured by plaque assay on tk-143 cells.

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We have determined the exact nature of two thermosensitive (ts) adenovirus mutants, H5ts19 and H5ts149, which map to different genes in the E2 transcription unit. The H5ts19 mutation appears to stem from a single base-pair change of A-T to G-C at position 1840 (numbering as in ref. 1), corresponding to codon 154 of the gene coding for DBP. This results in a glutamine-to-arginine change in the amino-terminal domain of the protein. H5ts19 is defective in a late stage of infection, during virus assembly. This phenotype strongly differs from that described for the limited number of known DBP mutants, indicating that DBP is not only functional during DNA replication, but also plays a role in the late phase of the infection cycle. The defect of the (N group) mutant H5ts149 affects the initiation of viral DNA replication. Marker rescue experiments followed by nucleotide sequence analysis of H5ts149 DNA revealed a single point mutation in the gene coding for the Ad pol. A transition of C-G to A-T at position 7563 (numbering as in ref. 2) changes amino acid residue 411 of Ad pol, a leucine residue, to phenylalanine. This mutation is located in a region conserved among various DNA polymerases, which suggests an important role of this domain in DNA replication.