Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation

Abstract

The specific mechanisms of charged polymer modulation of retrovirus transduction were analyzed by characterizing their effects on virus transport and adsorption. From a standard colloidal perspective two mechanisms, charge shielding and virus aggregation, can potentially account for the experimentally observed changes in adsorption behavior and biophysical parameters due to charged polymers. Experimental testing revealed that both mechanisms could be at work depending on the characteristics of the cationic polymer. All cationic polymers enhanced adsorption and transduction via charge shielding; however, only polymers greater than 15 kDa in size were capable of enhancing these processes via the virus aggregation mechanism, explaining the higher efficiency enhancement of the high molecular weight molecules. The role of anionic polymers was also characterized and they were found to inhibit transduction via sequestration of cationic polymers, thereby preventing charge shielding and virus aggregation. Taken together, these findings suggest the basis for a revised physical model of virus transport that incorporates electrostatic interactions through both virus-cell repulsive and attractive interactions, as well as the aggregation state of the virus.

FIGURE 1

Polybrene's enhancement of retrovirus transduction and adsorption is saturable, and is not due to toxicity or limiting concentrations of virus. (A) Ecotropic virus was supplemented with concentrations of polybrene between 0 and 28 μg/ml. NIH-3T3 cells were exposed to the virus samples for 48 h, lysed, and β-galactosidase levels were measured using an established ONPG-based assay. (B) An ecotropic virus stock (solid circles), as well as 1:2 (open circles) and 1:4 (solid squares) dilutions of the stock in fresh medium, were supplemented with between 0 and 12 μg/ml polybrene. Confluent NIH-3T3 cells were exposed to the same virus samples for 2 h, lysed, and levels of cell-associated p30 were measured via ELISA. (C) NIH-3T3 cells were plated in a 96-well plate at 5000 cells/well. After 24 h, the medium was removed and then replaced with fresh medium with concentrations of polybrene between 0 and 64 μg/ml. After 48 h, the relative cell density was measured via Orange G assay.

FIGURE 2

Poly-L-lysine's enhancement of virus transduction is size dependent. (A) Ecotropic virus was supplemented with concentrations of 1–4-kDa (solid circles), 4–15-kDa (open circles), or 15–30-kDa (solid squares) poly-L-lysine. NIH-3T3 cells were exposed to the virus samples for 48 h, and transduction was measured via the ONPG assay. (B) Ecotropic virus was supplemented with concentrations of 30–70-kDa (solid circles), 70–150-kDa (open circles), 150–300-kDa (solid squares), or >300-kDa (open squares) poly-L-lysine. NIH-3T3 cells were exposed to the virus samples for 48 h, and transduction was measured via the ONPG assay.

FIGURE 3

Poly-L-lysine's enhancement of virus adsorption is size dependent and parallels its effect on transduction. Ecotropic virus was supplemented with 4 μg/ml of each molecular weight poly-L-lysine. (A) NIH-3T3 cells were exposed to the virus samples, and after 48 h, β-galactosidase activity was measured via the ONPG assay. (B) Confluent NIH-3T3 cells were exposed to the same samples of virus for 2 h, and adsorption was measured via p30 ELISA.

FIGURE 4

Adsorption of FITC-labeled poly-L-lysine on target cells is rapid, dose dependent, and independent of cell type. (A) Confluent NIH-3T3 cells were trypsinized and pelleted via centrifugation for 5 min at 100 × g, 4°C. The cell pellets were resuspended in concentrations of FITC-labeled, 50-kDa mean molecular mass poly-L-lysine between 0 and 100 μg/ml, then incubated at 37°C on a rotary shaker. After 1 h, the cells were pelleted, washed with 1% FBS/PBS, resuspended in ice-cold 1% FBS/PBS, and analyzed via flow cytometry. (B) NIH-3T3 cells prepared as described above were exposed to 40 μg/ml 50-kDa mean molecular mass PLL for various times then analyzed via flow cytometry. (C) Confluent NIH-3T3 (solid circles) and CHO (open circles) cells prepared as described above were exposed to various concentrations of 50-kDa mean molecular mass poly-L-lysine for 1 h, then analyzed via flow cytometry.

FIGURE 5

Chondroitin sulfate proteoglycan abolishes FITC-labeled poly-L-lysine adsorption to target cells. Confluent NIH-3T3 cells were trypsinized and pelleted via centrifugation for 5 min at 100 × g, 4°C. The cell pellets were resuspended in 1% FBS/PBS containing 40 μg/ml FITC-labeled poly-L-lysine (open bars) or 40 μg/ml poly-L-lysine plus 40 μg/ml chondroitin sulfate proteoglycan (solid bars), then incubated at 37°C on a rotary shaker. After 1 h, the cells were pelleted, washed with 1% FBS/PBS, resuspended in ice-cold 1% FBS/PBS, and analyzed via flow cytometry.

FIGURE 6

Cationic polymers neutralize the negative ζ-potential on the virus and target cell. (A) Confluent NIH-3T3 cells were trypsinized and pelleted via centrifugation at 100 × g for 5 min, 4°C. The cell pellet was resuspended in isotonic PBS and 10⁶ cells were placed into a cuvette containing 2 ml isotonic PBS. The cell suspension was supplemented with optimal transduction enhancing concentrations of the various cationic polymers (i.e., poly-L-lysine and polybrene), and was incubated at 37°C. After 2 h, the ζ-potential of the cells was measured via phase analysis light scattering using a ZetaPALS instrument. The dashed line represents the baseline cell surface ζ-potential of −21.73 ± 1.08 mV. (B) Ecotropic virus was pelleted via ultracentrifugation for 1.5 h, at 275,000 × g, 4°C. The virus pellet was resuspended in isotonic PBS at one-tenth the original volume, and 20 μl was added to a ZetaPALS cuvette containing 2 ml isotonic PBS. The virus suspension was supplemented with optimal transduction enhancing concentrations of the various cationic polymers (i.e., poly-L-lysine and polybrene), and was incubated at 37°C. After 2 h, the ζ-potential of the cells was measured via phase analysis light scattering using a ZetaPALS instrument. The dashed line represents the baseline virus surface ζ-potential of −13.24 ± 0.68 mV.

FIGURE 7

Cationic polymer-mediated aggregation of retrovirus particles is size dependent. Ecotropic retrovirus was concentrated via ultracentrifugation at 275,000 × g for 1.5 h at 4°C, and the resulting virus pellet was resuspended in one-tenth the original volume of isotonic PBS. The concentrated virus was supplemented with optimal transduction enhancing concentrations of the cationic polymers (poly-L-lysine and polybrene) and incubated at 37°C for 1 h. The effective particle diameter was measured via laser light scattering in a ZetaPALS instrument. The dashed line represents the diameter of an untreated virus sample (101.3 nm).

FIGURE 8

A revised physical model of retrovirus transport. Electrostatic interactions among the virus, target cell, and charged polymers determine the nature and magnitude of the driving force for virus adsorption. All cationic polymers are capable of enhancing adsorption via neutralization of negative cell and virus surface charges; however, only large molecular weight polymers can aggregate virus sufficiently to enhance adsorption via sedimentation. Anionic polymers inhibit the processes of adsorption and transduction via sequestration of cationic polymers, preventing charge shielding and virus aggregation.