Expression of complement regulatory protein on porcine endogenous retrovirus (PERV) depends on molecular size

Expression of complement regulatory proteins (CRP) on pig cells is an effective means to avoid hyperacute rejection. However, pig endogenous retrovirus (PERV) from pig cells transfected with CRP may acquire resistance to human serum (HS). The present study investigated the size limitations of the transfected CRP that can be easily expressed and function on PERV particles. cDNAs of various sized DAF(CD55)s, including single-, double-, triple-, tetra-, as well as 2.1- and 2.2-DAF, were prepared. Pig endothelial cells (PEC) were transduced with the LacZ gene, and were then infected with PERV-B to produce PEC(Z)/PB. The extent of complement-mediated lysis by the transfectant molecules on PEC(Z)/PB was then determined. HEK293 cells were incubated with PEC(Z)/PB culture supernatants in the presence of HS and the LacZ pseudo-type assay was then carried out. Amelioration of complement-mediated lysis by the hybrid molecules was verified in each PEC(Z)/PB clone. All molecules appeared to effectively protect xenogeneic cells against complement-mediated lysis. While PERVs from the PEC(Z)/PB with both the single-DAF and double-DAF were resistant to HS, PERVs from the triple-DAF and tetra-DAF showed no significant increase in resistance. In addition, the PERVs from PEC(Z)/PB with 2.1-DAF and 2.2-DAF were less resistant than PEC with double-DAF. Resistance to HS was steadily attenuated with increasing size of the DAF molecule. The resistance to HS was disappeared by the anti-DAF blocking mAb, indicating that PERVs from the transfectants express DAF molecules on the surface of the PERV. The data clearly indicate that, to avoid the induction of resistance to HS in PERV particles, relatively large CRPs, such as triple-DAF and tetra-DAF or DAF with other large molecules, should be employed in the production of transgenic pigs.