Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant

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The adenovirus Ad5(pymT) has been used to express middle T antigen at very high levels in 293 cells. The middle T antigen produced was localized to membranes and was modified in the same way as that expressed in polyoma virus-infected mouse cells. It was phosphorylated in vivo on serine residues and in vitro on tyrosine residues. The in vivo phosphorylations occurred between residues 223 and 275. The middle T antigen encoded by A d5(pymT) was phosphorylated in vitro in a complex with human pp60jCSrC. Interestingly, the extreme overexpression of middle T antigen did not cause a parallel increase in the amount of complex; most of the pp60C src remained unassociated. Immunoaffinity purification resulted in approximately 100 ,ug of middle T antigen from a 100-mm tissue culture dish. Several cell proteins copurified with the Ad5(pymT)-derived middle T antigen. Two of these, the 74-and 63-kilodalton species, are of particular interest because they were also purified from mouse tumors expressing middle T antigen.

“…Adenovirus stocks were prepared on 293 cells grown in Dulbecco modified Eagle medium with 10% fetal calf serum. Ad5(pymT) and Ad5(309) are described in the accompanying paper (1).…”

Section: Methodsmentioning

confidence: 99%

“…A major obstacle to the elucidation of its action has been the absence of a rich source of protein. The hybrid adenovirus-polyomavirus Ad5(pymT) just described (1) provides such a source. However, the value of this Ad5(pymT) vector or any expression system depends upon how similar the protein being expressed is to that found in its original environment.…”

mentioning

confidence: 99%

Characterization of middle T antigen expressed by using an adenovirus expression system

Schaffhausen

Bockus

Berkner

et al. 1987

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“…For several years, human adenoviruses (Ads) have been used as vectors for expression of foreign genes in mammalian cells (1,2,5,8,11,16,19,20,25,29,32,48, 50, 51, 57, 58) and studied for potential use in live viral vaccines (7,9,13,20,26,28,35,37,(39)(40)(41)57) and, more recently, for gene therapy (27,34,36,42,43,45,46,52,53,56). A number of properties of the Ad system make it a good candidate for each of these applications, not the least of which is the extensive understanding of the structure and biology of Ads that has been gained through their use as a model system to study DNA replication, transcription, RNA processing, and protein synthesis (17).…”

mentioning

confidence: 99%

Packaging capacity and stability of human adenovirus type 5 vectors

Bett

Prevec

Graham

1993

409

129

Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, * Corresponding author.

“…In addition, there are several examples in which Ad vectors were used to express biologically functional proteins. The SV40 large-T antigen expressed from an Ad vector retains its DNA replication functions (47), and the polyomavirus middle-T antigen also retains its biological functions when expressed from an Ad vector (3,12,39). Because basal and EIAinduced expression of the CAT gene from the Ad EII-early promoter was not hampered by the presence of small-t (Fig.…”

Section: Discussionmentioning

confidence: 99%

Simian virus 40 small-t does not transactivate RNA polymerase II promoters in virus infections

Rajan

Dhamankar

Rundell

et al. 1991

Transcriptional stimulatory properties of virus-encoded transactivators appear to be critical for viral gene expression and may be linked to cellular transformation in certain cases. Recently, the simian virus 40 (SV40) 17-kDa small-t antigen was shown to stimulate transcription of polymerase II and III genes in transient transfection assays. In experiments performed in our laboratory, two of the polymerase II promoters of the adenovirus genome, namely, the ElI-early and EIII promoters, were transactivated by the small-t antigen in transient transfection assays. To further elucidate the mechanism of this transactivation, we examined the ability of small-t to transactivate the adenovirus type 5 EIl-early and EIII promoters in CV-1 cells under conditions in which the small-t gene or the reporter genes were introduced into the cells through transfection and other routes. In one approach, we used established CV-1 cell lines which constitutively express the small-t gene, and study of the EII-early promoter was afforded by infection of an EIA-negative adenovirus type 5 variant. For the second approach, a recombinant adenovirus was constructed in which small-t was expressed from a replication origin-negative SV40 early promoter in the EIA region of an adenovirus vector (Ad-SV-t). The effect of small-t on adenovirus EII-early and EIII promoter expression was studied in coinfection or single-infection experiments. In both cases, transcription of the adenovirus early promoters was not stimulated by small-t. These and other results indicate that transactivation of polymerase II promoters by small-t occurs only when the target gene is in a transiently transfected state. Thus, small-t-mediated transactivation of polymerase II promoters is dependent on the type of assay system used and may be mechanistically different from that of the widely studied EIA.