Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction

Abstract

The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed.

FIG. 1

Visualization of MLV vector and HIV-1 particles. Virus supernatants of MLV vectors bearing MLV-A Env (ampho) and no Env and HIV-1 GUN-1 isolate were air dried and fixed with 4% paraformaldehyde on glass slides. After permeabilization, immunostaining was performed with anti-CA antibodies followed by FITC-labeled secondary antibodies (anti-CA) and with anti-SU antibodies followed by Texas red-labeled secondary antibodies (anti-SU). Images for green and red fluorescence were acquired separately and overlaid (merged). Bar, 2 μm.

FIG. 2

Estimation of size and physical number of MLV particles. A LacZ(MLV) supernatant was filtered through 450-, 200-, 100-, and 20-nm-pore-size filters in succession. Red-fluorescent microspheres were added to the supernatant before filtration (top panels) or after filtration (bottom panels) at 2.7 × 10⁸/ml and processed for immunostaining for MLV CA proteins as in Fig. 1. Stained viral particles and fluorescent microspheres from the sample filtered before microsphere addition (bottom panel) were counted in five random fields of 5,500 μm², and the microsphere/viral particle ratio was measured. Results are expressed as mean values of the estimated ratios ± standard errors of the mean. LacZ titer was measured on TE671 cells. Bar, 2 μm. n.d., not determined.

FIG. 3

Negative staining and immuno-EM of viral particles. A mixture of virus particles and beads was adsorbed to an EM nickel grid by ultracentrifugation (A) or by a 1-h incubation at 37°C in the presence of 8 μg of Polybrene per ml (B). Samples were then processed for capsid immunostaining and negative staining with PTA (A) or uranyl acetate (B). The beads measure 110 nm in diameter. Bar, 100 nm.

FIG. 4

Binding of MLV vector particles to TE671 cells. Cell supernatant of TE671 cells producing LacZ(MLV-A) (A), LacZ(MLV-E) (B), LacZ pseudotype without Env proteins (C), and no virus particle (D) was added to TE671 cells. The cells were washed extensively after a 1-h incubation and then fixed with acetone-methanol (1:1). Samples were stained for MLV CA proteins with FITC-labeled secondary antibodies. Bar, 5 μm.

FIG. 5

Comparison of FACS and confocal microscopy analyses of MLV vector binding to TE671 cells. Suspended TE671 cells were incubated with supernatants of TE671 cells producing LacZ(MLV-A) (ampho), LacZ(MLV-E) (eco), LacZ pseudotype without Env proteins (no Env), and no virus particle (no virus) for 1 h, washed, and then fixed with 4% paraformaldehyde. The cells were stained with either anti-MLV CA (anti-CA) antibodies after permeabilization or anti-MLV SU antibodies without permeabilization. The micrographs are projected optical sections performed every 1 μm perpendicular to the z axis throughout the sample. Bar, 5 μm. Histograms of cells incubated with vector particles are shaded and shown together with histograms of cells incubated with virus-free cell supernatant (white).

FIG. 6

Kinetics of binding of MLV vector particles to adherent cells. TE671 cells were incubated with MLV vector particles with MLV-A Env (ampho) and without any Env protein (no Env) for 1, 5, 15, and 30 min and then processed for immunostaining with anti-MLV CA antibodies followed by FITC-labeled secondary antibodies. Bar, 5 μm.

FIG. 7

Kinetics of MLV vector particles dissociation from cells. TE671 cells were incubated with MLV vector particles with MLV-A Env (ampho) and without any Env protein (no Env). After 1 h, the cells were washed and kept in fresh medium at 37°C and 0.1% sodium azide (A) or in PBS at 4°C (B) for the indicated time. Samples were then processed for capsid immunostaining. The micrographs are projected optical sections performed every 1 μm. Bar, 5 μm.

FIG. 8

Binding of MLV vector particles to different adherent cells. Cell cultures were incubated with MLV-A (ampho), MLV Env defective (no Env), or mock viral supernatants (no virus). After a 1-h incubation, the cells were washed, fixed, permeabilized, and stained for capsid proteins. Bar, 5 μm.

FIG. 9

Binding of MLV vector particles and MLV-A Env to suspension cells. Adherent TE671 cells harvested with EDTA and three suspension cultures were incubated with MLV-A particles (ampho), MLV Env-defective particles (no Env), or mock supernatant (no virus). (A) Microscopy analyses. Cells were permeabilized and stained for MLV-CA. Bar, 10 μm. (B) FACS analysis. Cells were stained for SU and analyzed by FACS. White and black histograms show samples incubated with Env-defective and MLV-A supernatants, respectively. Micrographs are projected optical sections performed every 0.5 μm.