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

Faruk Sinangil, Abraham Loyter, D. J. Volsky*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)88-92
Number of pages5
JournalFEBS Letters
Volume239
Issue number1
DOIs
StatePublished - 24 Oct 1988

Keywords

  • Fluorescence dequenching
  • HIV
  • Membrane fusion
  • Target cell membrane

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