A newly cloned pig dolichyl-phosphate mannosyl-transferase for preventing the transmission of porcine endogenous retrovirus to human cells

Abstract

Porcine endogenous retrovirus (PERV) is a major problem associated with successful clinical xenotransplantation. In our previous study, reducing the high mannose type of N-glycan content proved to be very effective in downregulating PERV infectivity. In this study, dolichyl-phosphate mannosyltransferase (D-P-M), an enzyme related to the early stages of N-linked sugar synthesis was studied. The pig cDNA of the encoding D-P-M was newly isolated. The RNA interference (siRNA) for the D-P-M was applied and transfected to PEC(Z)/PB cells, a pig endothelial cell line with the Lac Z gene and PERV-B, to reduce the levels of high mannose type N-glycans. Compared with the mock line, the temporary PEC(Z)/PB lines showed a decreased mRNA expression for pig D-P-M, and each line then showed a clear destruction of PERV infectivity to human cells in the Lac Z pseudotype assay. The PEC(Z)/PB was next transfected with pSXGH-siRNA, H1-RNA gene promoter. The established PEC(Z)/PB clones with pSXGH-siRNA clearly led to the downregulation of PERV infectivity, as evidenced by the decreased levels of the mRNA for pig D-P-M. Reducing D-P-M enzyme activity represents a potentially useful approach to address the problem of PERV infections in clinical xenotransplantations.

Xenotransplantation literature update: April-May, 2010

“…Reduction of the high mannose type of N-glycan content can decrease the infectivity of porcine endogenous retrovirus (PERV). Yamamoto et al [14] studied dolichyl-phosphate mannosyltransferase (D-P-M), an enzyme related to the early stages of N-linked sugar synthesis. siRNA for D-P-M was transfected into a pig endothelial cell line with the Lac Z gene and PERV-B to reduce the levels of high mannose type N-glycans.…”

“…Yamamoto et al. [14] studied dolichyl‐phosphate mannosyltransferase (D‐P‐M), an enzyme related to the early stages of N‐linked sugar synthesis. siRNA for D‐P‐M was transfected into a pig endothelial cell line with the Lac Z gene and PERV‐B to reduce the levels of high mannose type N ‐glycans.…”

Methods and Tools for Detection and Evaluation of the Risks of Porcine Endogenous Retrovirus in Porcine to Human Xenotransplantation

“…They have shown that by essentially altering the mannose residues on the N-linked glycan moieties of PERV envelope, they demonstrate a decrease in PERV infectivity titers on human cells in vitro (Miyagawa, Nakatsu et al 2006) (Yamamoto, Nakatsu et al 2010). …”