The location of v-src in a retrovirus vector determines whether the virus is toxic or transforming

We prepared infectious stocks of an avian retrovirus, a modified spleen necrosis virus, containing the herpes simplex virus type 1 thymidine kinase gene and the avian sarcoma virus v-src gene. Viruses were recovered after cotransfection of chicken cells with DNA of recombinants between cloned spleen necrosis virus thymidine kinase and v-src and with DNA of cloned reticuloendotheliosis virus strain A. When v-src was inserted near the 5'end of the viral genome, only low titers of recombinant virus were recovered. Most of the recovered viruses were smaller than expected and did not transform the morphology of rat or chicken cells. A very small amount of virus of the expected structure was recovered; this virus transformed rat cells and expressed v-src. Cotransfection data indicated that one reason we failed to recover a significant titer of recombinant virus is that efficient expression of v-src is acutely toxic to chicken and dog cells. Insertion of v-src near the 3' end of the viral genome, such that it was expressed at a lower level compared with the 5'-v-src-containing virus, yielded a higher titer of recombinant virus, and this virus was transforming. The differences in the recovery and transforming activity of these viruses indicate that the location of an oncogene in the viral genome is an important factor regulating the level of its expression and whether or not this expression is toxic or transforming to cells.