Construction and Preliminary Characterization of a Library of “Lethal” Preterminal Protein Mutant Adenoviruses

Abstract

Adenoviruses containing lethal in-frame insertion mutant alleles of the preterminal protein (pTP) gene were constructed with cell lines that express pTP. Thirty in-frame insertion mutant alleles, including 26 alleles previously characterized as lethal and 4 newly constructed mutant alleles, were introduced into the viral chromosome in place of the wild-type pTP gene. The viruses were tested for ability to form plaques at 37°C in HeLa-pTP cells and at 32°C and 39.5°C in HeLa cells. Two of the newly constructed viruses exhibited temperature sensitivity for plaque formation, one virus did not form plaques in the absence of complementation, seven additional mutants exhibited a greater than 10-fold reduction in plaque formation in the absence of complementation, and another eight mutants exhibited stronger phenotypes than did previously characterized in-frame insertion mutants in the plaque assay. These mutant viruses offer promise for analysis of pTP functions.

Role of conserved residues in the activity of adenovirus preterminal protein

“…Previous attempts to make mutations in pTP have been confined to linker insertion mutagenesis and deletion of residues from the N terminus of pTP (Fredman et al ., 1991 ; Freimuth & Ginsberg, 1986 ; Pettit et al ., 1989 ; Roovers et al ., 1991 , 1993 ; Schaack et al ., 1999 ). Introduction of such mutations into the virus genome and transfections into a pTP complementing cell line allowed the growth of virus containing these mutations.…”

Adenovirus vectors deleted for genes essential for viral DNA replication

“…In contrast, Ad mutants ts for pTP function were not discovered in the random screens. However, ts pTP mutant viruses were constructed after two and four amino acid insertion mutagenesis (41)(42)(43)(44).…”