The function(s) provided by the adenovirus-specified, DNA-binding protein required for viral late gene expression is independent of the role of the protein in viral DNA replication

Abstract

The adenovirus type 2 (Ad2) host range mutant Ad2hr400 grows efficiently in cultured monkey cells at 37°C, but is cold sensitive for plaque formation and late gene expression at 32.5°C. After nitrous acid mutagenesis of an Ad2hr400 stock, cold-resistant variants were selected in CV1 monkey cells at 32.5°C. One such variant, Ad2ts400, was also temperature sensitive (ts) for growth in both CV, and HeLa cells. Marker rescue analysis has been used to show that the two phenotypes, cold resistant and temperature sensitive, are due to two independent mutations, each of which resides in a different segment of the gene encoding the 72-kilodalton DNA binding protein (DBP). The cold-resistant mutation (map coordinates 63.6 to 66) is a host range alteration that enhances the ability of the virus to express late genes and grow productively in monkey cells at 32.5°C. The temperature-sensitive mutation is in the same complementation group and maps to the same segment of the DBP gene (map coordinates 61.3 to 63.6) as the well-characterized DBP mutant Ad5tsl25. Like Ad5tsl25, Ad2ts400 is unable to replicate viral DNA or to properly shut off early mRNA expression at the nonpermissive temperature. Two sets of experiments with Ad2ts400 suggest that DBP contains separate functional domains. First, when CV1 cells are coinfected at the nonpermissive temperature with Ad2 plus Ad2ts400 (Ad2 allows DNA replication and entry into, but not completion of, the late phase of infection), normal late gene expression and productive growth occur. Second, temperature shift experiments show that, although DNA replication is severely restricted at the nonpermissive temperature in ts400-infected monkey cells, late gene expression occurs normally. These results indicate that the DBP activity required for normal late gene expression in monkey cells is functional even when the DBP's DNA replication activity is disrupted.

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis

“…Although adenoviruses cannot grow productively in monkey cells, adenovirus mutants capable of productive infection of monkey cells can be isolated (3,36,38). The lesions in these host range mutants map to the 72-kilodalton DNA-binding protein and are located at a site in the polypeptide distant from the mutation sites of mutants affecting the replication and transcriptional regulation activities of the polypeptide (53). Thus, for both SV40 and the human adenoviruses, a separate domain of a major DNAbinding and regulatory polypeptide is involved in permitting the virus to grow productively in monkey cells.…”

Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein

“…Analysis of adenovirus DNA synthesis and preparation of low-molecular-weight DNA from adenovirus-infected cells have been described previously (40). To compare the numbers of adenovirus templates in infected cells, isolated nuclei remaining after cytoplasmic RNA extraction (see below) were lysed in 3.8 ml of 50 mM Tris (pH 7.4)-10 mM EDTA-100 mM NaCI-0.5% SDS, and then 0.2 ml of 10 mg of pronase per ml was added.…”

“…The early region 2A gene (E2A) of human adenovirus types 2 and 5 (Ad2 and Ad5, respectively) encodes a 72-kilodalton (kDa) protein which possesses single-stranded DNA binding activity (34,46). This polypeptide, commonly called the adenovirus DNA-binding protein (DBP), is thought to contain two physical (25, 43) as well as functional (4,31,40) domains. A large number of functions have been ascribed to DBP.…”

“…DBP differs from the gene products encoded by ElA and ElB, however, in that it is probably required in very large amounts (up to 107 molecules per cell) duhing a viral infection. Although the gmDBP cell lines complement the growth of E2A ts mutants (27), the ts mutations probably inactivate only a subset of the functions of DBP (40). Thus it was unclear whether these cell lines would allow the isolation of DBP-negative adenovirus mutants.…”

Identification of two nuclear subclasses of the adenovirus type 5-encoded DNA-binding protein

“…The 44-kDa carboxyl-terminal fragment can bind single-stranded DNA (21) and can substitute for intact DBP in an in vitro DNA replication system (4). The 26-kDa amino-terminal fragment, which contains most of the protein's phosphorylation sites (21), retains its activity(ies) required for normal late gene expression even when the DBP DNA replication function is perturbed by ts mutations in the carboxyl terminus (37). Together these results suggest that DBP contains at least two functionally and physically separable domains.…”