Xenogeneic cell therapies and xenotransplantation, i.e., the therapeutic use of living cells, tissues, and organs from animals, show some promise to alleviate the limited supply of allografts in the treatment of human disorders. Pigs are the donor species of choice (15), especially since strategies to overcome rejection have been developed (17,47,48,53). Recently, pigs in which the alpha-1,3-galactosyltransferase locus has been genetically knocked out were generated (10, 24).The existence of porcine endogenous retroviruses (PERV), which are germ line transmitted (43), and of porcine DNA viruses that can persist without symptoms in their natural host (e.g., herpesviruses) (13) strengthened objections to the clinical use of pig xenografts due to the possible generation of xenozoonosis (15,54,55).PERV display approximately 50 proviral integration sites in the pig genome (1, 43). Virions observed in cell lines are morphologically related to C-type viruses (2). Genetically, three classes of PERV (classes A, B, and C), which differ in their env genes, are known (25). Recent reports demonstrated that PERV which are released from different pig cell lines are able to infect human cells in vitro (32,43,58,59). In a retrospective study, no cross-species transmission of PERV in 160 patients treated with pig tissue was observed (41). On the other hand, in an NOD/SCID mouse model, the diabetic and immunodeficient animals showed infection with and expression of PERV in different tissues after xenotransplantation of porcine islet cells, suggesting that PERV are xenoreactive in vivo (57).Even though a subspecies of miniature swine that failed to produce PERV was recently identified (38), the emergence of hitherto-undescribed PERV genomes (29,36,42) shows how difficult a comprehensive risk assessment will be.As methods for the direct quantification of PERV are poorly developed (44), we have established a real-time PCR to determine the PERV proviral load in either porcine or infected human cells as well as a real-time one-step reverse transcription-PCR (RT-PCR) to quantify the virion release in cell culture supernatants. Recently, we demonstrated that PERV long terminal repeats (LTRs) are characterized by significant promoter activities which are linked to the existence of a repeat box in U3 representing an enhancer (49). To prove the binding of transcription factors to the 39-bp repeats, we performed electrophoretic mobility shift assays (EMSA) and supershift assays. By generation of modified LTRs lacking the repeat box, U3, R, or U5, we investigated whether further elements exist which regulate the transcriptional machinery. In regard to the significance of PERV for xenotransplantation, we investigated whether immunosuppressive drugs such as cyclosporine (CysA) or prednisolone (Pred) have any impact on the promoter activity.
MATERIALS AND METHODSQuantification of proviral load by real-time PCR. The genomic DNAs of the porcine cell line PK15, the human cell line 293 productively infected with PERV derived from PK15 cells (293/PER...