7th International AIDS Conference Florence, Italy — June 16-21, 1991

Int Conf AIDS 1991 Jun 16-21; 7:99 (abstract no. M.A.1031)
Zeira M, Littman D, Volsky DJ; Molecular Virology Laboratory, St. Luke's/Roosevelt Hospital Center, Columbia University, New York, N.Y.

OBJECTIVE: To establish parameters of HIV-1 entry into cells by measuring fusion between viral envelopes and CD4+ T cell lines or freshly isolated PBLs, and to correlate between viral entry and expression in these cells and in mouse cells expressing human CD4 receptors.

METHODS: Sucrose gradient purified HIV-1 CAT virions were labeled with octadecylrhodamine B-chloride and incubated with target cells. Virus-cell membrane fusion was measured as the increase in the fluorescence signal (dequenching, DQ). Expression of HIV-1 immediately after entry was determined by PCR and CAT assays, and by intracellular p24 and HIV-1 RNA assays several days later. Target cells included PBLs, human T lymphoid A2.01 cells expressing either CD4 or CD8 receptors, and mouse 3T3 fibroblasts and thymoma 1010 cells, stably expressing human CD4 receptors (conferred by transfection).

RESULTS: DQ values observed following incubation of human CD4+ cells with labeled virus were dependent on cell number (max DQ at 2x10(6) cells) temperature (max DQ at 37 degrees C and none at 4 degress C) and incubation time (50% of DQ within 20 min and 100% DQ within 30 min.). Preincubation of purified labeled virus with sCD4 resulted in sCD4 dose dependent inhibition of DQ. This showed that the fusion event, as measured by DQ, depends upon specific gp120-CD4 receptor binding. No difference of fusion values was observed between resting PBLs and PHA stimulated PBLs. Stable expression of CD4 converted mouse 3T3 fibroblasts and thymoma 1010 cell lines from negative to positive DQ with HIV-1. The extent of fusion observed in CD4+ murine cells was comparable to CD4+ human cells. That DQ correlates with viral entry in these cells is being tested by intracellular CAT activity or by detection of HIV-1 DNA by PCR.

CONCLUSIONS: The DQ technique enables us to determine quantitatively the viral and cellular parameters required for viral entry, and to correlate entry and expression in human and animal cells.

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