Figure 1.
Measurement of infectious HIV-1 virions by blue-cell counting in TZM-bl cells.
(A) and (B), representative images of blue TZM-bl cells in 12-well plates for quantitation of infectious particles showing well-resolved single cells and clusters of cells such as doublets. (C) Fraction of single or clustered blue cells depends on the presence of Vpr in single-cycle HIV-1 virions. Vpr+ virions were generated from transfection of 293T cells with 1 μg pNL4-3E- and 0.1 μg pcDNA3.1REC. Vpr− virions were generated from transfection of 293T cells with 1 μg pNL4-3R−E− and 0.1 μg pcDNA3.1REC. Both virions were harvested 48 hours post transfection without media change. TZM-bl cells were infected by either of these two virions and the resulting single or clustered blue cells were counted. Error bars are standard deviations from six independent replicates of the same experiments. (D) The number of blue cells was plotted as a function of virion dilution factor per 100 μl of virus. The red line shows its linear regression. The virus was produced by transfection of 293T cells using 1.0 μg pNL4-3E− plasmid and 0.2 μg pcDNA3.1REC, followed by collection 48 hours post transfection without media change. Error bars are standard deviations from six independent replicates of the same experiments.
Figure 2.
Luciferase activity assay to monitor infection of TZM-bl cells by single-cycle HIV-1 virions.
The RLUs measured with a microplate reader were plotted as a function of input Ci.p. on a double-logarithmic scale. It shows saturation behavior when Ci.p. exceeds 106/ml, and shows excess noise when Ci.p. is below 103/ml (inset). Error bars are standard deviations from three independent replicates of the same experiments.
Figure 3.
Confocal imaging of EGFP-Vpr virions and particle quantitation.
(A) A representative fluorescence image of EGFP-Vpr virions deposited on a coverslip surface. The image is 1024 by 1024 pixels with a physical size of 124 by 122 nm in x and y dimensions. (B) Distribution of virion fluorescence intensity identified from the confocal image using a custom-written MATLAB script. (C) The correlation between numbers of fluorescent particles identified from a FOV and the input p24 values measured using ELISA. The linear regression is shown in red line. Error bars for each sample are standard deviations from at least nine different areas analyzed on a coverslip surface.
Figure 4.
Ci.p., Cp.p., and infectivity of HIV-1 virions as a function of harvest time post transfection.
Filled circles are for virions produced using 1.0 μg pNL4-3E− plasmid and 0.2 μg pcDNA3.1REC. Open circles are for Gag particles produced using 1.0 μg pNL4-3E− plasmid only. The dashed line is (C) is fit of the time course to a sum of two-exponential kinetics. Error bars are standard deviations from three independent replicates of the same experiments.
Figure 5.
Kinetics of HIV-1 virion infectivity decay.
Time course of HIV-1 virion spontaneous inactivation was measured in either 12-well plate (triangle) and fitted using single exponential decay (dashed line), or in 1.5 ml test tube (circles) and fitted using single exponential decay (solid line). Error bars are standard deviations from three independent replicates of the same experiments.
Figure 6.
Effect of media change post transfection on HIV-1 virion infectivity.
The virus was produced by transfection of 293T cells using 1.0 μg pNL4-3E− plasmid and 0.2 μg pcDNA3.1REC, and virions were collected at 18 and 48 hours post transfection. The differences in infectivity were confirmed by the Paired-sample t-test. Error bars are standard deviations from five independent replicates of the same experiments.
Figure 7.
Effect of media change on HIV-1 virion infectivity tagged with EGFPs.
Panels (A), (B) and (C) show the infectivity, Ci.p. and Cp.p. of HIV-1 virions tagged with EGFP-Vpr, as a function of harvest time post transfection, with media change six hours post transfection (filled circles) and without (open circles). Panel (D) shows the infectivity of free EGFP virions as a function of the time of media change post transfection. The virus was collected 24 hours post transfection. Error bars are standard deviations from three independent replicates of the same experiments.
Figure 8.
Infectivity of EGFP-tagged single-cycle virions as a function of input EGFP.
(A) Infectivity of EGFP-Vpr virions as a function of input pEGFP-Vpr plasmid. The viruses were collected 48 hours post transfection without media change. (B) Infectivity of free EGFP virions as a function of the fraction of EGFP-tagged mutant provirus in a total of 1 μg mutant provirus. The virions were collected 24 hours post transfection without media change. Error bars are standard deviations from three independent replicates of the same experiments.
Figure 9.
Two-color flow cytometry to quantitate fraction of cells that carried reporter plasmids.
(A) Negative control in the absence of both plasmids, which was used to gate cell populations in transfected cells. (B) 293T cells transfected with 0.6 μg pEGFP-Vpr and 0.2 μg pmCherry-Vpr in 2-ml volume in a 35-mm dish. The gates established based on negative control were used to quantitate fractions of cells that carry EGFP only, mCherry only and both fluorescent proteins in a mixed populations, with the percentages of each population indicated in the cytogram. (C) 293T cells transfected with 0.6 μg pEGFP-Vpr and 0.2 μg pmCherry-Vpr, with a media change six hours post transfection; (D) 293T cells transfected with 0.2 μg pEGFP-Vpr and 0.6 μg pmCherry-Vpr; (E) 293T cells transfected with 0.2 μg pEGFP-Vpr and 0.6 μg pmCherry-Vpr, with a media change six hours post transfection. All cell samples were collected at 48 hours post transfection. 488 nm and 560 nm lasers were used for excitation of EGFP and mCherry respectively. Two independent analyses yielded results that were identical within errors.
Figure 10.
Dependence of HIV-1 virion infectivity on envelope plasmid input during transfection.
(A), (B) and (C) show the infectivity, Ci.p. and Cp.p. for virions produced with various inputs of envelope plasmid pcDNA3.1REC. Circles and triangles are for virions collected 18 and 48 hours post transfection, respectively, with filled symbols representing samples with media change and open symbols representing samples without media change. Error bars are standard deviations from three independent replicates of the same experiments, except for 48 hours without media change (open triangle dashed line), which were duplicates.
Figure 11.
Western blotting of virion and cell lysate samples collected from cells transfected under various conditions.
The amount of envelope plasmid in μg is indicated above the lanes. The double bands from anti-gp120 staining corresponded to gp120 (bottom) and gp160 (top), respectively. β-actin expression was also probed simultaneously to validate the comparison across different transfection conditions. Two independent analyses yielded results that support the same conclusions.
Figure 12.
Infection assay in Rev-CEM cells for various single-cycle HIV-1 virions.
(A) Negative control in the absence of infection, which was used to gate cell populations in infected cells. (B) A representative cytogram for Rev-CEM cells infected by single-cycle virions generated with 2 μg envelope. The virions were harvested 18 hours post transfection with media change six hours post transfection. (C) and (D) show Ci.p. and infectivity in Rev-CEM cells for virions produced with various inputs of envelope plasmid pcDNA3.1REC. Circles and triangles are for virions collected 18 and 48 hours post transfection, respectively, with filled symbols representing samples with media change and open symbols representing samples without media change. Error bars are standard deviations from three independent replicates of the same experiments.