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Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching
Author(s) -
Sinangil Faruk,
Loyter Abraham,
Volsky D.J.
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)80551-9
Subject(s) - sendai virus , cell fusion , chemistry , centrifugation , incubation , lipid bilayer fusion , membrane , fluorescence , glutaraldehyde , microbiology and biotechnology , biophysics , virus , chromatography , biochemistry , biology , virology , cell , physics , quantum mechanics
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 NH 4 Cl 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.