Therapeutic effect of RANTES-KDEL on inhibition of HIV-1 in CD34(+) human hematopoietic stem cells (hHSC).
Cell surface receptors, such as the CCR5 chemokine receptors, represent key determinants of the human immunodeficiency virus type 1 (HIV-1) entry into target cells. The CC-chemokine, RANTES (regulated upon activation, normal T-cell expressed and secreted), a ligand for CCR5, have been targeted to the lumen of endocytoplasmic reticulum (ER) using a KDEL (ER-retention signal) fusion termed RANTES-KDEL and this construct was found to prevent effectively transport of newly synthesized CCR5 to the cell surface. Lentiviral vectors have emerged as potent and versatile tools of gene transfer for basic and applied research are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. For this reason, an HIV-based lentiviral vector expressing RANTES-KDEL, pLenti6/V5-R-K, was constructed and then cotransfected with the ViraPower Packaging Mix (pLP1, pLP2, and pLP/VSVG) into 293FT cells to produce a replication-incompetent lentivirus stock. The lentiviral stock was titrated using HeLa cells, and the expression of the gene of interest, RANTES, was detected by indirect immunofluorescence. Based on the above results, the lentiviral stock was transduced into CD34(+) human hematopoietic stem cells (hHSC) separated magnetically from the cord blood (the purity was 96.8% evaluated by flow cytometry). Finally, the levels of p24 in the cultures of pLenti6/V5-R-K-transduced CD34(+) hHSC were detected after infection by HIV-1 DP1 (a R5-tropic HIV-1 strain, which was isolated by the Centers for Disease Control and Prevention of China in Henan province in 2000 from a Chinese man who had asymptomatic HIV-1 infection with a history of blood transfusions). It was shown that pLenti6/V5-R-K transduction inhibited expression of the DP1 p24 antigen by 51%, 58% and 60% on the 4th, 7th and 10th day respectively (P<0.05).
Sun
Li
L
Center of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
Zhang
Ying
Y
Huang
Chang-Xing
CX
Qu
Xiao-Li
XL
Zhang
Ye
Y
Zhang
Jiu-Cong
JC
Wei
Xin
X
Zhuang
Yan
Y
Zhai
Song
S
Peng
Mei-Juan
MJ
Jiang
Hong
H
Hao
Chun-Qiu
CQ
Li
Yan-Hong
YH
Lu
Jiang
J
Lian
Jian-Qi
JQ
Sun
Yong-Tao
YT
Bai
Xue-Fan
XF
D018952:Antigens, CD34 / Q000032:analysis*; D001483:Base Sequence; D065100:CCR5 Receptor Antagonists; D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000235:genetics / Q000276:immunology*; D002681:China; D004721:Endoplasmic Reticulum; D016655:HIV Core Protein p24 / Q000096:biosynthesis; D015497:HIV-1 / Q000276:immunology*; D006367:HeLa Cells; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000821:virology*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D021381:Protein Transport
D018952:Antigens, CD34; D065100:CCR5 Receptor Antagonists; D018946:Chemokine CCL5; D016655:HIV Core Protein p24