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

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.