New reporter cell lines to study macrophage-tropic HIV envelope protein-mediated cell-cell fusion.
The infection of human cells by HIV-1 virus can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCR5), and HIV ADAgpl60 as fusion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCR5 with CHO cells expressing HIV ADAgpl60, a 300-fold increase in luciferase activity could be elicited relative to control. No luciferase activity was detected when HXB2gpl60 (T-tropic) was used instead of ADAgpl60 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contrast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was detected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green fluorescent protein (GFP) was used as the reporter instead of luciferase. In combination with fluorescence microscopy, the GFP reporter system allowed visualization of the fusion events in real time. Compared with previously described HIV fusion models, this system has several advantages, including simplicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easily adapted to study other HIV fusion events.
Hong
Y L
YL
Parke-Davis Pharmaceutical Research, Division of the Warner-Lambert Company, Ann Arbor, Michigan 48105, USA.
Wu
L H
LH
Cui
M
M
McMaster
G
G
Hunt
S W
SW
Chung
F Z
FZ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015704:CD4 Antigens / Q000502:physiology*; D016466:CHO Cells; D002459:Cell Fusion / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D019302:HIV Envelope Protein gp160 / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008264:Macrophages / Q000821:virology*; D008856:Microscopy, Fluorescence; D019713:Receptors, CCR5 / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D019302:HIV Envelope Protein gp160; D008164:Luminescent Proteins; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases