Isolation and characterization of a viable adenovirus mutant defective in nuclear transport of the DNA-binding protein

Abstract

The isolation and characterization of an adenovirus mutant, Ad5dl802r1, containing two independent deletions in the 72-kilodalton (kDa) DNA-binding protein (DBP) gene is described. The two deletions remove amino acids 23 through 105 of DBP, resulting in the production of a 50-kDa product. Expression of this truncated DBP was delayed 12 to 24 h compared with that of the 72-kDa protein produced by wild-type adenovirus type 5. The DBP was located primarily in the cytoplasm of infected cells, whereas the wild-type product was predominantly nuclear. Therefore, DBP appears to contain a nuclear localization signal within the deleted region. Ad5dl802r1 DNA synthesis, viral late gene expression, and virus production were all delayed 12 to 24 h and were approximately 10-fold lower than with wild-type adenovirus type 5. These phenotypic properties can be accounted for by the delay in synthesis and the inefficient accumulation of the 50-kDa DBP within the nucleus of infected cells. The truncated DBP also lacks the majority of amino acids which are phosphorylated in the normal protein. The loss of these phosphorylation sites does not appear to seriously impair the ability of the protein to carry out its functions.