Synchronized infection of cell cultures by magnetically controlled virus

Abstract

To override the diffusion-limited adsorption step of viral infection, we magnetically synchronized cell attachment. Human immunodeficiency virus type 1-based lentivirus preparations were rendered magnetically reactive by association with magnetite nanoparticles, 50 nm in diameter. Application of a magnetic field resulted in immediate redistribution of the viral inoculum to the cell-associated state and completion of the productive adsorption process within 1 min. Independent of adsorption time, viral concentration, and diffusion rate, infection subsequently progressed by the receptor-mediated entry mechanism. Synchronization of this rate-limiting step of infection may now be applied to analyze isolated events in the viral replication sequence.

FIG. 1.

Magnetically controlled adsorption of lentivirus to RAEC monolayers. (A) Redistribution of viral particles from the medium to the cell-associated state. Preparations of lenti-VSVG virus expressing β-galactosidase (8 ng of p24 antigen) were preincubated with MNPs or PBS and added to RAEC monolayers (4 × 10⁵ cells per well) with or without the application of a magnetic field. After a 7-min incubation period at 37°C and 5% CO₂, the infection medium was removed, and cells were washed three times with culture medium. Both fractions were then assayed using the HIV-1 p24 antigen capture assay kit (SAIC Frederick, AIDS Vaccine Program). Values represent the mean p24 antigen levels ± standard errors (SE) (error bars) in an experiment performed with four replicate samples. (B) Infection by lenti-VSVG expressing luciferase. Cultures were adsorbed with the virus for the indicated periods, washed three times with culture medium, and incubated for 48 h until luciferase enzyme activity was assayed using the dual luciferase kit (Promega Corp., Madison, Wis.). Light emission was measured with the Lucy-1 microplate luminometer (AnthosLabtec Instruments, Salzburg, Austria) as previously described (4). Protein content in each preparation was determined by the Bradford method, and results are presented as the mean relative light unit (RLU) measurement per microgram of protein extracted ± SE (error bars). (C) Infection by lenti-VSVG expressing β-galactosidase (3 × 10³ IU). Values represent the mean number of β-galactosidase-positive cells ± SE (error bars) counted after 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) staining of cultures 48 h postadsorption (1).

FIG. 2.

Adsorption kinetics of lenti-VSVG virus. RAEC monolayers (4 × 10⁵ cells per well) were adsorbed with the lenti-VSVG virus expressing β-galactosidase (3 × 10³ IU in 600 μl of culture medium). At the indicated time points, the infection medium was removed and added to an adjacent well similarly plated with RAECs. Cells initially adsorbed were then washed three times with culture medium, and all cultures were incubated further until X-Gal staining was performed 48 h later. Data are presented as the number of β-galactosidase-positive cells counted in cultures initially adsorbed with the virus (A) and in cultures incubated with the residual virus-containing medium (B). Each datum point represents the mean value obtained from three or four replicate samples ± SE (error bars).

FIG. 3.

Effect of viral concentration on infection. RAEC monolayers (4 × 10⁵ cells per well) were adsorbed with a fixed inoculum of lenti-VSVG virus expressing β-galactosidase (2 × 10⁴ IU per well) in increasing volumes of culture medium. After the indicated incubation period at 37°C and 5% CO₂, nonadsorbed virus was removed by three washes with culture medium, and samples were incubated further until X-Gal staining 48 h later. Data represent the mean number of β-galactosidase-positive cells ± SE (error bars) from three separate experiments performed in triplicate and are expressed as a percentage of the number of positive cells counted at the lowest dilution.