Simian retrovirus vectors for gene transfer in nonhuman primate cells

We have recently identified and sequenced a molecular clone of the serogroup 2 simian retrovirus (SRV), D2/RHE/OR/V1, that retains an enhanced ability to infect specific T cell lines. In this report, using deletion mutagenesis, we localized the psi packaging signal, necessary for packaging of D2/RHE/OR/V1 particles, to the genomic region 345-650, which comprises the 5' intergenic region (IR) and the extreme 5' portion of the gag gene. To build an SRV-based gene transfer system and to reduce the possibility of recombination and regeneration of replication-competent viruses, we constructed split-genome D2/RHE/OR/V1 plasmid recombinants containing distinct and non-overlapping retroviral gene regions and several replacement components. For the retrovirus gene transfer vehicle, we deleted the D2/RHE/OR/V1 structural genes and substituted a cassette including the psi-packaging region, the beta-galactosidase reporter gene, and the 3' IR. Both packaging cell recombinants were used to generate stable monkey packaging cell lines; the gene transfer vehicle was subsequently transfected into the packaging cell lines, and replication-defective viruses were recovered for subsequent infection into fresh monkey cells. Successful infection by the recovered viruses verifies the potential efficacy of the SRV-based system as a research tool for gene transfer of heterologous genes into nonhuman primate cells.