Differential human serum-mediated neutralization of PERV released from pig cells transfected with variants of hDAF.
BACKGROUND
Expression of complement regulatory proteins (CRP) on pig endothelial cells (PEC) is an effective means of avoiding induction of hyperacute rejection by human sera. However, pig endogenous retrovirus (PERV) from PEC transfected with CRP may acquire resistance to human sera. This study investigated a form of transfected CRP that is easily expressed on PERV particles.


METHODS
The PEC line was transfected with the Lac Z gene and PERV-B to investigate PERV infectivity using a Lac Z pseudo-type assay. The cDNAs of several modified DAF (CD55) were then transfected into the PEC(Lac Z)/P-B lines using lipofection. DAF expression was verified by FACS analysis. Complement-dependent PEC lysis was tested to verify the complement regulatory function of the expressed DAF. HEK293 cells were incubated with PEC culture supernatants with or without human sera. The inoculated 293 cells were histochemically stained and Lac Z-positive blue foci were counted. The rate of reduction in Lac Z-positive cells resulting from the addition of human serum was then calculated. In addition, to assess the localization of the expressed DAF, flotation sucrose density analysis was performed.


RESULTS
While PERV released from PEC expressing delta-short consensus repeat 2 (delta-SCR2) DAF (lacking CRP function) showed no change in resistance to human serum compared to control cells, PERV from cells expressing delta-SCR1 DAF (with CRP function) showed a significant increase in resistance. The DAF-blocking antibody assay indicated that PERV from the DAF transfectants expressed DAF molecules on the surface of the retrovirus. While delta-SCR1 DAF (PI-anchor form) significantly inhibited the reduction of Lac Z-positive cells by human serum, the reduction of Lac Z-positive cells by human serum was less inhibited in the case of transmembrane (TM)-types of DAF-HLA-G, modified influenza hemagglutinin (HA) and MCP (delta-CYT form). However, the reduction in each TM-type DAF was slightly less than that observed in naive and mock cells. The flotation sucrose density analysis of these transfectants indicated that the PI-anchor form of DAF is a raft-associated protein, and most TM-types of DAF are non-raft proteins.


CONCLUSIONS
Induction of resistance to human serum in PERV, depends on the form of the CRP tail. The CRP/TM hybrid that does not associate with lipid rafts, is a suitable form of CRP for gene transduction.
Okura
Eiji
E
Division of Organ Transplantation, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
Ishimaru
Aiko
A
Yamamoto
Aki
A
Nakatsu
Shino
S
Shirakura
Ryota
R
Okabe
Masaru
M
Sawa
Yoshiki
Y
Fukuzawa
Masahiro
M
Okumura
Meinoshin
M
Miyagawa
Shuji
S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000097:blood / Q000276:immunology; D018958:CD55 Antigens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D012190:Retroviridae / Q000276:immunology*; D044967:Serum / Q000276:immunology*; D013552:Swine / Q000235:genetics / Q000276:immunology* / Q000821:virology*; D014162:Transfection
D000906:Antibodies; D018958:CD55 Antigens