Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene.
Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.
Sakamoto
Tatsunori
T
Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Ushijima
Hiroshi
H
Okitsu
Shoko
S
Suzuki
Eiko
E
Sakai
Koji
K
Morikawa
Shigeru
S
Müller
Werner E G
WE
D001681:Biological Assay; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism*; D002459:Cell Fusion; D017930:Genes, Reporter; D015726:Giant Cells; D019302:HIV Envelope Protein gp160 / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D015704:CD4 Antigens; D019302:HIV Envelope Protein gp160; D019718:Receptors, CXCR4