FIG. 4.
Clusters of CARex-Fc-Ad2 and ts1 are phagocytosed and released into THP-1 cells. (A) CARex-Fc dose-dependent stimulation of fluid-phase uptake by Fcγ-R-targeted Ad2 and ts1. THP-1 cells (5 × 105) were incubated with different ratios of preincubated CARex-Fc dimers to Ad2 (8 μg) in the cold for 60 min, washed, warmed for 5 min, pulsed with dextran-FITC for 5 min, harvested, and analyzed by FACS. Results are expressed as levels of stimulation of dextran uptake compared with uninfected cells. The experiment was performed twice with similar results. (B and D) Ad2 (B) and ts1 (D) in complex with CARex-Fc are delivered to the THP-1 cytosol. Ad2 and ts1 were incubated with 300 and 30 CARex-Fc dimers/particle, respectively, and then incubated with THP-1 cells at an MOT of 100 for 1 h in the cold. The cells were washed, warmed for 60 min, fixed, and processed for TEM. Arrows indicate endosomal virus aggregates, and arrowheads depict cytoplasmic virus particles. Bar = 500 nm. The numbers of virus particles at the plasma membrane (pm), endosomes (end), cytosol (cyt), and the nuclear membrane (nm) were quantified. Mean values derived from the indicated numbers of cells are expressed as percentages of total virus particles with corresponding SEM. n, number of Ad particles. The large asterisks indicate differences between endosomal Ad2 and endosomal ts1 with P values of <0.01 in one-sided t tests. The small asterisk indicates a difference in the cytosolic localization of ts1 in HeLa cells with a P value of 0.05 (C). The P values of the other ts1 compartments in comparison with Ad2 in THP-1 and ts1 in HeLa cells were >0.05, i.e., the differences are not significant. (C) ts1 predominantly localizes to endosomes of HeLa cells in the absence of CARex-Fc. HeLa cells expressing CAR and αv integrins were infected with ts1 for 60 min upon cold binding of virus, fixed, and processed for TEM. The subcellular distribution of virus particles was quantified. Less than 10% of the virus particles were at the plasma membrane, and none were found at the nuclear membrane (not shown).