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, Ad5dl802rl, 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. Ad5dl802rl 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 c,an 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.

Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis

“…The phenotypes of these mutants suggest that the overall phosphorylation state of this protein may be important for its expression. This result, together with results of several other early studies (16,53), argues that DBP enhances its own expression.…”

“…2). None of the mutants showed delayed growth, as was recently observed for H5dl802rl, a mutant which contains a large deletion in the amino-terminal domain that removes many of the phosphorylation sites (16). Substitution of one or two sites (H5pt801 through H5pt804) had little effect on the final virus yield.…”

“…In a parallel experiment to that shown in Fig. 3, the levels of DBP which had accumulated by 16 h postinfection in the presence of hydroxyurea were determined by immunoblotting by using SDS-PAGE (on 10% gels), a rabbit polyclonal anti-DBP serum, and '25I-labeled protein A (10 uCi). Lanes 1 through 7 correspond to the protein extracts from H5pt801through H5pt807-infected cells, and lane 8 represents the wt AdS control.…”

Imaging the adenovirus infection cycle

“…Currently, DBP is frequently used as reference stain to describe spatial and temporal events in the nucleus of Ad‐infected cells . In addition, DBP was used to characterize Ad replication itself to identify cellular and viral proteins that are sequestered into viral RCs or used as spatial or timing reference to study non‐RC‐linked events . Recent super‐resolution microscopy performed on isolated replication compartments from infected HeLa cells suggests a dynamic RC subcompartmentalization of DBP .…”

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis

“…DBP is essential for DNA replication (for reviews, see Chase and Williams, 1986; Hay et al ., 1995; van der Vliet, 1995) and is involved in many steps in the viral life cycle, like transformation (Ginsberg et al ., 1974), transcriptional control (e.g. Cleghon et al ., 1989; Zijderveld et al ., 1994) and assembly of the virus (Nicolas et al ., 1983). DBP binds cooperatively to single‐stranded DNA (ssDNA) in a non‐sequence‐specific fashion.…”