Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen.

Abstract

Adenovirus-polyomavirus recombinant viruses were constructed in vitro by inserting a hybrid transcription unit composed of the adenovirus type 2 major late promoter and the early coding region of polyomavirus into the adenovirus type 5 vector Ad5AE1/d1309. The vector lacks the Ela and Elb transcription units and contains a unique restriction endonuclease cleavage site in their place. The polyomavirus genomic insert contained a small deletion which precluded the synthesis of functional small and middle T antigen but allowed for the synthesis of large T antigen. One recombinant virus, Ad5PyR39, which contained the hybrid transcription unit in the opposite transcriptional orientation from the overall direction of late-gene transcription, was studied in detail. Ad5PyR39 replicated efficiently without a helper virus in human 293 cells and expressed hybrid mRNAs of the expected size and composition that were translated to yield large T antigen. The large T antigen synthesized in 293 cells was the same size as that produced in mouse 3T6 cells lytically infected with polyomavirus, and this protein bound efficiently and specifically to the large-T-antigen-binding sites in polyomavirus DNA. Moreover, the large T antigen encoded by the recombinant virus proved capable of catalyzing the replication in mouse 3T6 cells of a plasmid containing the polyomavirus origin for DNA replication. Comparison of the amount of large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with Ad5PyR39, under optimal conditions for each system, revealed at least a fivefold greater yield of the protein on a per cell basis in the latter system compared with the former. Ad5PyR39 should prove to be useful to isolate large quantities of functional polyomavirus large T antigen for structural and biochemical studies.

Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes.

“…The uptake studies of [3H]ASOR were performed by incubating the protein with the hepatocytes for the specified period oftime, after which the medium containing the protein was removed, the cells were washed with PBS-(phosphatebuffered saline, minus Ca2+ and minus Mg2+), 1 Preparation of Adenovirus. Adenovirus (d1312) was received as a gift from T. Shenk (Princeton University, Princeton) and grown in 293 cells and purified by banding twice in CsCl gradients (24). The concentration of the virus was determined by UV spectrophotometric analysis, and the virus was stored in 10% (vol/vol) glycerol at -20°C.…”

DNA template effect on RNA splicing: two copies of the same gene in the same nucleus are processed differently.

“…The Ad-SVR1 12 adenovirus SV40 recombinant contains the SV40 early region replacing the adenovirus E1A and ElB regions. An analogous virus AdS-PyR39 containing the polyoma virus early genes substituted for much of the ElA and ElB genes was obtained from John Hassell (Massie et al, 1986). An analogous virus AdS-PyR39 containing the polyoma virus early genes substituted for much of the ElA and ElB genes was obtained from John Hassell (Massie et al, 1986).…”

Adenovirus-mediated gene transfer into striated muscles

“…Over the last two decades substantial data have accumulated regarding the biology and molecular characteristics of adenoviruses. This wealth of information promoted their use as expression vectors initially for recombinant protein production [11,20,21,34,54,82,93,103,138,174], then as recombinant vaccines [10,15,36,71,81,101,106], and more recently as gene transfer agents in animal experiments [143,146,150] as well as in human trials [32,40,124,149].…”