Abstract:
OBJECTIVES: To gain insight into initial steps of entry of HIV into cells. METHODS: Membrane fusion of HIV-1 with CD4+ cells was measured by spectrofluorometry and infection assays. HIVenv-mediated cell fusion and syncytia formation is monitored by image enhanced fluorescence video microscopy following infection of cells with recombinant vaccinia. Fluorescently-labeled plasma membrane vesicles containing CD4 were constructed by hypotonic lysis of cells. Measurement of the kinetics of association of sCD4 with cell-surface associated gp120 was performed by means of flow cytometry or infection assays. RESULTS: The interaction of CD4 with gp120/41 sets a complex set of events in motion which finally terminates in the delivery of the HIV-1 nucleocapsid into the cell. Using a variety of virological, cell biological and biophysical techniques we have quantified a temporal sequence of those events, which include binding, conformational changes, formation of fusion junctions, and coalescence of membranes and aqueous spaces. The trigger was examined by studying interactions of sCD4 with gp120/41. We found that HIV-1 infectivity was dependent on cell density of the suspensions, and at high cell concentrations even very high concentrations of sCD4 were inefficient in blocking virus infection. The role of the target membrane in formation of fusion junctions was examined by studying fusion of gp120-gp41+ effector cells with hybrid giant cells from monkey CD4+ cells and CD4- human cells. The role of the lymphocyte adhesion molecule LFA-1 on the various stages of the process was examined using LFA-1- cell lines. We also examined plasma membrane vesicles derived from CD4 cells as a new tool to study gp120-gp41-mediated membrane fusion, and inhibition of HIV-1 infection. CONCLUSIONS: The quantitative information gathered about CD4-gp120/41 leading to the merging of membranes will further our understanding of mechanisms of viral entry.
Keywords: Animal Cell Fusion Cell Membrane *Cytopathogenic Effect, Viral CD4-Positive T-Lymphocytes/*MICROBIOLOGY Haplorhini Human Hybrid Cells/MICROBIOLOGY HIV Envelope Protein gp120/*PHYSIOLOGY HIV Envelope Protein gp41/*PHYSIOLOGY HIV-1/*PHYSIOLOGY Image Enhancement Lymphocyte Function-Associated Antigen-1/*PHYSIOLOGY *Membrane Fusion Microscopy, Fluorescence Videotape Recording ABSTRACT 921230
M92C5346
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