A newly cloned pig dolichyl-phosphate mannosyl-transferase for preventing the transmission of porcine endogenous retrovirus to human cells.
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.
Yamamoto
Aki
A
Division of Organ Transplantation, Department of Surgery, and Osaka University Graduate School of Medicine, Japan.
Nakatsu
Shino
S
Kondo
Akihiro
A
Asato
Tetsuya
T
Okabe
Masaru
M
Fukuzawa
Masahiro
M
Miyagawa
Shuji
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D020077:Endogenous Retroviruses / Q000378:metabolism*; D006801:Humans; D007763:Lac Operon; D008364:Mannosyltransferases / Q000235:genetics* / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000378:metabolism; D013552:Swine; D014183:Transplantation, Heterologous / Q000379:methods*; D014777:Virus Diseases / Q000517:prevention & control*
D034741:RNA, Small Interfering; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase