Article Figures & Data
Figures
- Fig. 1.
Flow diagram of spinoculation; see the text for details. Cx, time in culture.
- Fig. 2.
Spinoculation enhances HIV-1 viral adsorption to CEM-SS cells and RT to similar extents. The numbers of copies of viral RNA and cDNA (extended first strand) per cell were measured immediately after challenge of 2 × 105 cells with 100 μl of a 3.0-μg/ml p24gag stock of HIV-1IIIB at 1 × g or 1,200 × g using kinetic PCR. The numbers of copies of viral cDNA per cell were also measured after 5 h in culture. At 24 h, total levels of p24gag (cell associated and supernatant) were determined by ELISA and converted to the number of viral particles produced per cell. UD, undetectable.
- Fig. 3.
Immunofluorescence analysis of CEM-SS cells infected at 1,200 × g (A) or 1 × g (B). Twenty-four hours after challenge, cells were fixed and stained with a p24gag-specific antiserum followed by a fluorescein isothiocyanate-labeled secondary antibody. Cells were also stained with 4′,6′-diamidino-2-phenylindole and visualized at a magnification of ×400.
- Fig. 4.
The modest g forces used for spinoculation sediment HIV-1 particles. One-hundred-microliter aliquots of an HIV-1 viral stock were exposed to 1 × g or 1,200 × g for 2 h in a 96-well tissue culture microtiter plate at room temperature in the absence of cells. Serial 20-μl aliquots were then withdrawn from the upper meniscus and assayed for the presence of p24gag by ELISA. The data shown represent the means of three independent sample sets.
- Fig. 5.
Spinoculation-mediated virus binding is CD4 and temperature dependent. CEM-SS cells were challenged at 1 × g or 1,200 × g for 2 h under different conditions, and the levels of cell-associated p24gag were determined immediately by ELISA and converted to numbers of virion equivalents. Control cells were inoculated in the presence of 10 μg of murine IgG1/ml at 25°C; the murine anti-CD4 IgG1 monoclonal antibody was present at a concentration of 10 μg/ml; polybrene was added to a final concentration of 8 μg/ml; temperature was maintained at 4°C; or cells were vortexed continuously during virus challenge (in this case, cell viability was shown to be maintained for the following 24 h). ND indicates that a particular combination of conditions was not tested.
- Fig. 6.
Spinoculation enhances virus adsorption to different T cells and with VSV G pseudotypes. (A) The numbers of cell-associated virion equivalents following challenges of CEM-SS, HUT78, SupT1, or primary CD4-positive T cells were calculated as for Fig. 5. (B) Similarly, the effects of spinoculation were compared for HIV-1IIIB and HIV-1/Δenv (VSV G) pseudotypes.
