Figure 1

Production of retroviruses by conventional packaging cell transcription or in vitro transcribed genomes. A) Retroviruses are produced by transfecting vector plasmids into a packaging cell line (e.g. 293T-based Phoenix cells) that expresses requisite viral structural proteins. Cis-sequences in the LTR promote transcription and editing of the retroviral RNA genome which is then exported to the cytoplasm for assembly into a viral particle. Mature virions bud from the surface and can infect target cells. B) If an intron is embedded in the retroviral sequence it is removed by the nuclear splicing prior to packaging, such that the packaged retroviral genome lacks the intron. C) A retroviral vector incorporating an additional polyadenylation signal (pA) will yield an unproductive truncated viral genome. D) A new approach is to synthesize the retroviral genomic transcript in vitro and transfect it into the packaging cell cytoplasm where it is directly packaged into a virus particle. In the pT system vector, the promoter/enhancer (U3) sequence in the 5'LTR is replaced with the promoter sequence for bacteriophage T7 RNA polymerase, enabling in vitro transcription of the retroviral genomic RNA. Avoidance of nuclear functions allows packaging of retroviral RNA incorporating various editing signals (introns and polyadenylation signals) and subsequent transfer into target cells.