During the past year we have initiated an investigation into the regulation of retrotransposition in mammalian cells. The experimental model is the mouse Fv-1 gene which dominantly restricts certain preintegration steps and possibly the integration step involved in the replication cycle of retroviruses. Retrovirus vectors with selectable markers but no viral structural genes are used in these experiments. These constructs are not capable of replication and are transferred to recipient cells by packaging into retrovirus virions. Colony formation of mouse fibroblasts in the presence of the antibiotic G418 is the basis of our assay system. This is superior in many ways to assay systems involving replication competent retroviruses. By rescue of the marker neo resistance with molecularly cloned retrovirus with known sensitivities to various Fv-1 alleles, we have established that the integration of the retrovirus vector is subject to restriction by the Fv-1 gene. Our results indicate that non Fv-1 related differences in sensitivity to retrovirus infection of certain cell lines may be due to the traditional assay system (XC plaque assay) and are less of a problem with the G418 resistant colony assay. Recombinant DNA constructs have been prepared to create helper virus free packaging cell lines with N-, B-, and NR- tropism. This will allow us to analyze the role of two hit kinetics and abrogation in the mechanism of retrotransposon restriction by the Fv-1 gene product.
