Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching

FEBS Letters

Loyter, Abraham²  Volsky, D.J.¹  Sinangil, Faruk¹ 

[1] Molecular Virology Laboratory, St. Luke's/Roosevelt Hospital Center and Columbia University, New York, NY 10019, USA;Dept. of Biochemistry, Hebrew University, Jerusalem, Israel

关键词: HIV;    Fluorescence dequenching;    Membrane fusion;    Target cell membrane;    HIV;    human immunodeficiency virus;    SV;    Sendai virus;    AIDS;    acquired immunodeficiency syndrome;    DQ;    membrane fluorescence dequenching;    R-18;    octadecylrhodamine-B chloride;   

DOI  :  10.1016/0014-5793(88)80551-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Human immunodeficiency virus (HIV) was purified by sucrose gradient centrifugation and labeled with octadecylrhodamine B-chloride (R-18) under conditions resulting in 90% quenching of the fluorescence label. Incubation of R-18-labeled HIV (R-18/HIV) with CD4-positive CEM and HUT-102 cells, but not with CD4-negative MLA-144 cells, resulted in fluorescence dequenching (DQ, increase in fluorescence) of 20–25%. Similar level of DQ was observed upon incubation of CEM cells with R-18-labeled Sendai virus. DQ was observed when R-18/HIV was incubated with CD4⁺ cells at 37°C, but not at 4°C. Most of the increase in fluorescence occurred within 5 min of incubation at 37°C and was independent of medium pH over the range of pH 5–8. Preincubation of cells with the lysosomotropic agent NH₄Cl had no inhibitory effect on DQ. Complete inhibition was observed when target cells were fixed with glutaraldehyde prior to R-18/HIV addition. Our results demonstrate application of membrane fluorescence dequenching method to a quantitative measurement of fusion between HIV and target cell membranes. As determined by DQ, HIV penetrates into target cells by a rapid, pH-independent, receptor-mediated and specific process of fusion between viral envelope and cell plasma membrane, quite similar to that observed with Sendai virus.

【 授权许可】

Unknown