Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

Abstract

The DNA-bindihg protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central iole in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by IBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DB3P is totic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50-to 200-fold, and within 8 h its synthesis fromb the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.A number of multifunctional proteins in euearyotes have been defined by,a combination of genetic and biochemical studies. One of the best characterized of these is the simian virus 40 (SV40) large T antigen, which exhibits separate functional domains as discerned by mapping of sets of mutations which alter one but not another of the various protein functions.The human adenovirus 72-kilodalton DNA-binding protein (DBP) also plays several independent roles during the infectious cycle of this virus. Several mutants with temperature sensitive (tsj alterations in the DBP have been indispensable in defining several of the functions of this protein. The ts alterations at the nonpermissive temperature diminish the single-strand, DNA-binding activity of the protein in vitro (52) and inhibit DNA replication in vivo (13, 47, 53) and in vitro (20). Normal turn-off of viral early genes during the late phase of the lytic cycle is also disrupted at the nonpermissive temperature in cells infected with the ts mutant (6,9,10,37,38). The DBP directly affects early gene turn-off as well as DNA replication, since inhibition of viral DNA synthesis with either drugs or ts lesions in other viral early genes does not affect early gene expression (9, 10). In addition, at the nonpermissive temperatui*, the ts mutants transform rat cells in culture with an enhanced frequency compared with wild-type (wt) virus (16,54).A second class of mutants which greatly enhance the ability of this human virus to grow in monkey cells defines yet a second set of functions of DBP (1,26,29). In monkey cells infected with wt human adenovirus, the viral early genes are properly expressed (3), and viral DNA replication occurs no...

Induction of apoptosis and G2/M arrest by infection with replication-deficient adenovirus at high multiplicity of infection

“…8,25 Toxicity in cell culture has been reported for the E2a gene product. 26,27 We examined expression of the E4 ORF6 gene and were able to detect expression of this gene in all cell lines at MOIs leading to 100% or 400% gene transfer. Despite deletion of the essential E1a and E1b genes, residual expression of cytotoxic genes apparently takes place.…”

Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.

“…In this category, the MMTV LTR promoter has been used to regulate expression of various oncogenes, including the middle T antigen of polyomavirus (47), v-ras (28), v-mos (46,56), and v-src (31), and recently to express gG of HSV-2 (54). A third application, used in this study, is the induced expression of genes whose products are cytotoxic (33). The MMTV LTR promoter has certain advantages over other inducible promoters, such as heat shock or metallothionein.…”

Development and application of adenoviral vectors for gene therapy of cancer

“…As a genetic system for the isolation and propagation of Ad mutants containing lesions in the E2A region, stably transfected HeLa cell lines were developed that express an integrated copy of the gene encoding the multifunctional Ad-DNA-binding protein (DBP) under control of a dexamethasone-inducible promoter. 188 With these cells (gmDBP) several novel E2A mutant Ads (Ad5 dl801 through Ad5 dl805) were generated through mutagenization of a cloned E2A gene in vitro and introduction of the mutated E2A into the viral genome by in vivo recombination. 189 Because these cells did not allow severely defective viruses to form plaques, additional gmDBP cell lines were constructed that allowed all DBP-negative mutants to form plaques.…”