During the past year we have continued our development of a model for regulation of retrotransposition in mammalian cells based on the Fv-1 gene restriction system. Specifically, we have engineered a cell line which packages retrovirus vectors in an Fv- 1 N tropic virion. The Fv-1 target molecule present in the virion results in an inhibition of certain preintegration steps during viral DNA synthesis in cultures of mouse cells with the Fv-1bb allele. The model system involves retrovirus vectors which are unable to replicate, thus providing a measure of a single round of successful reverse transcription and integration. This system will allow an analysis of the mechanism of the Fv-1 gene restriction previously unavailable. Biological activity of the Fv-1 packaging line has been evaluated by release of replication defective transforming virus or vectors containing a dominant selectable marker. Results indicate that the packaging construct has a propensity to regenerate infectious virus, most likely by recombination with the vector or endogenous retrovirus elements in the host mouse cell. New strategies are being developed to create packaging lines which are stable and unlikely to recover the ability to replicate autonomously. Fluorescent antibody assays and RNA blot hybridization analysis have demonstrated a low efficiency of viral gene expression in various packaging lines compared to virus infected cells. Attempts are being made to improve this by utilizing high efficiency transcription promoters. Recently we have employed oligonucleotide probes specific for the Fv-1 target determinant which allows a determination of Fv-1 tropism of endogenous retroviral elements. Preliminary results indicate that many endogenous nonecotropic proviral genomes contain the B- tropism specific sequence and are likely to be the source for generating B-tropic virus.
