[A non-infectious and quantitative cell-based bioassay for screening HIV entry inhibitors targeting HIV envelope proteins].
OBJECTIVE
To develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors.


METHODS
HL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method.


RESULTS
No syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner.


CONCLUSIONS
We have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.
Li
Min-min
MM
Center of Clinical Medicinal Laboratory, First Affiliated Hospital of Jinan University, Guangzhou 510630, China.
Xia
Cheng-lai
CL
Mao
Qin-chao
QC
Jiang
Shi-bo
SB
Liu
Shu-wen
SW
D001681:Biological Assay; D002459:Cell Fusion; D002460:Cell Line; D018920:Coculture Techniques; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015700:HIV Envelope Protein gp41 / Q000378:metabolism*; D023581:HIV Fusion Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D001616:beta-Galactosidase / Q000378:metabolism
D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D001616:beta-Galactosidase