A sensitive HIV-1 envelope induced fusion assay identifies fusion enhancement of thrombin.
To evaluate the interaction between HIV-1 envelope glycoprotein (Env) and target cell receptors, various cell-cell-fusion assays have been developed. In the present study, we established a novel fusion system. In this system, the expression of the sensitive reporter gene, firefly luciferase (FL) gene, in the target cells was used to evaluate cell fusion event. Simultaneously, constitutively expressed Renilla luciferase (RL) gene was used to monitor effector cell number and viability. FL gave a wider dynamic range than other known reporters and the introduction of RL made the assay accurate and reproducible. This system is especially beneficial for investigation of potential entry-influencing agents, for its power of ruling out the false inhibition or enhancement caused by the artificial cell-number variation. As a case study, we applied this fusion system to observe the effect of a serine protease, thrombin, on HIV Env-mediated cell-cell fusion and have found the fusion enhancement activity of thrombin over two R5-tropic HIV strains.
Cheng
De-Chun
DC
Department of Microbiology, Harbin Medical University, 194 Xuefu Road, Harbin, Heilongjiang 150081, China.
Zhong
Guo-Cai
GC
Su
Ju-Xiang
JX
Liu
Yan-Hong
YH
Li
Yan
Y
Wang
Jia-Ye
JY
Hattori
Toshio
T
Ling
Hong
H
Zhang
Feng-Min
FM
D002459:Cell Fusion / Q000379:methods; D002460:Cell Line; D019302:HIV Envelope Protein gp160 / Q000378:metabolism*; D023581:HIV Fusion Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D049410:Luciferases, Renilla / Q000096:biosynthesis; D008561:Membrane Fusion; D015728:Receptors, HIV / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D053586:Virus Internalization
D019302:HIV Envelope Protein gp160; D023581:HIV Fusion Inhibitors; D015728:Receptors, HIV; D049410:Luciferases, Renilla; D013917:Thrombin