Cytosolic Gag p24 as an index of productive entry of human immunodeficiency virus type 1

Abstract

We have investigated the cellular uptake of Gag p24 shortly after exposure of cells to human immunodeficiency virus (HIV) particles. In the absence of envelope glycoprotein on virions or of viral receptors or coreceptors at the cell surface, p24 was incorporated in intracellular vesicles but not detected in the cytosolic subcellular fraction. When appropriate envelope-receptor interactions could occur, the nonspecific vesicular uptake was still intense and cytosolic p24 represented 10 to 40% of total intracellular p24. The measurement of cytosolic p24 early after exposure to HIV type 1 is a reliable assay for investigating virus entry and early events leading to authentic cell infection.

FIG. 1

Confocal microscopy analysis of intracellular p24 after cell exposure to HIV-1. HeLa cells (middle panel) or P4 cells (right panel) were exposed to HIV-1_NL43 for 2 h at 4°C, washed, warmed to 37°C for 30 min, fixed, and labeled with anti-Gag MAbs. Left panel, control HeLa cells not exposed to HIV-1.

FIG. 2

p24 levels in subcellular extracts of cells exposed to HIV-1. HeLa, P4, or P4C5 cells were exposed to strain NL43 (lymphotropic) or JRCSF (macrophage tropic) or to HIV particles devoid of envelope protein (NL43Δenv), respectively, for 30 min at 4°C. Cells were extensively washed and were incubated at 37°C for 1 h. Cells were then treated with pronase, and p24 amounts in subcellular fractions were measured. Total intracellular p24 levels (in picograms) are indicated over the bars. The percentages of cytosolic and vesicular p24 are shown inside the bars. (a) Dose-response analysis for NL43; (b) analysis of the entry of HIV-1 strains with different tropisms (input, 300 ng of p24); (c) analysis of the entry in CEM cells (input, 400 ng of p24). Data are representative of at least three experiments.

FIG. 3

Effect of bafilomycin A₁ on HIV-1 infection efficiency and entry into P4 cells. Cells were either left untreated or treated with 0.5 μM bafilomycin A₁ during virus exposure. (a) Infection was assessed by measuring β-galactosidase activity in cell extracts. Viral input corresponded to 1 ng of p24. (b) Intracellular p24 levels were measured in the cytosolic and vesicular fractions. Total intracellular p24 levels (in picograms) are indicated over the bars. The percentages of cytosolic and vesicular p24 are shown inside the bars. Viral input corresponded to 300 ng of p24 during 1 h at 37°C. HIV/VSV is a VSV-G pseudotype of NL43. Data are from one of three representative experiments.