Using a series of single-stranded clones of adenovirus DNA, we determined the extent of RNA polymerase II transit early in infection for two rightward-reading transcription units. RNA synthesis beginning at the major late promoter (16.5 on the genomic map) continued until approximately 65 to 70 map units so that differential choices of mRNAs within that region were not based primarily on transcriptional decisions but rather on posttranscriptional decisions. Transcription from the major late promoter beginning at 16.5 map units, however, did greatly decrease before approximately 75 map units, ensuring that no mRNAs were formed with sequences beyond approximately 75 map units. Early transcription from E3 then began just past 75 map units (at a higher rate than transcription from the major late promoter); E3 transcripts terminated at least 2 kilobases downstream from a second and final poly(A) site in this transcription unit. The effectiveness of termination in E3 was greater than 95 to 99%.

Daily parenteral administration of exogenous interferon-gamma (IFN-gamma) induces or accelerates recovery in experimental and human infections. To develop an alternative delivery system, a replication-defective recombinant adenovirus expressing human IFN-gamma was constructed. The complete coding region of IFN-gamma was amplified by RT-PCR and inserted into an adenovirus cloning vector under the control of a human cytomegalovirus promoter. Recombinant adenovirus containing the IFN-gamma minigene (dAv-IFN-gamma) was isolated from 293 cells co-transfected with the linearized plasmid and an E1 region-deleted fragment of adenovirus genome. Following in vitro infection with dAv-IFN-gamma, dose-dependent and time-dependent expression of IFN-gamma, mRNA and production of soluble protein were demonstrated in human diploid fibroblat and HeLa cell cultures by Northern blot and ELISA, respectively. Extracellular protein secretion persisted for > = 4 weeks following initial transfection, and secreted IFN-gamma induced both antiviral activity (8000-25,000 U/ml) and macrophage activation with killing of intracellular Toxoplasma gondii and leishmania donovani. These results establish that dAv-IFN-gamma generates long-term secretion of biologically active IFN-gamma in vitro and suggest that this vector may be a useful delivery system for cytokine therapy.