Quantitative measurement of fusion between human immunodeficiency virus and cultured cells using membrane fluorescence dequenching.
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 degrees C, but not at 4 degrees C. Most of the increase in fluorescence occurred within 5 min of incubation at 37 degrees 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.
Sinangil
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Molecular Virology Laboratory, St. Luke's/Roosevelt Hospital Center and Columbia University, New York, NY 10019.
Loyter
A
A
Volsky
D J
DJ
D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D006678:HIV / Q000502:physiology*; D006801:Humans; D010222:Parainfluenza Virus 1, Human / Q000502:physiology; D011991:Receptors, Virus / Q000502:physiology*; D013050:Spectrometry, Fluorescence / Q000379:methods
D000945:Antigens, Differentiation, T-Lymphocyte; D011991:Receptors, Virus