Fig. 2
Step 3: 3D collagen limits HIV-1 particle diffusion and infectivity. a Representative confocal micrograph of fluorescent viral particles in media or within collagen. Fluorescent viral particles (green, HIVGFP or HIVmcherry) were incorporated into collagen (orange) or resuspended in medium overlaying the collagen matrix (suspension). Collagen was visualized using confocal reflection microscopy. Maximum projection of 10 µm stacks at 0.5 µm intervals is shown. Scale 5 µm. b Schematic overview of virion diffusion analysis. Motility of viral particles within collagen or in medium (suspension) was monitored by spinning disc confocal microscopy (×40 objective) recording images of one focal plane at maximum speed for 5 min (corresponding to 1475 frames; middle panel). c Viral particles were automatically tracked by single particle tracking (see Methods for details). Diffusion parameters were determined from mean square displacement (MSD) computed for at least 20,000 viral tracks (mean+/−SEM). d Determined anomalous diffusion exponent α, which is 1 for normal diffusion, scaled transport coefficient Г and diffusion coefficients D. e Space and time considerations for virus diffusion in suspension and collagen, taking into account the infectious half-life of HIV-1 of 17.9 h63. f Distribution of instantaneous velocities of single particles from tracks with durations between 0.8 and 15 s for virus in suspension (blue) or in collagen (brown). Boxed are number of tracking steps with velocities below 1 µm s−1. g Relative infectivity of HIV-1. Virus that had diffused out of collagen was tested for infectivity normalized for RT activity, using TZM-bl reporter cells in a blue cell assay. Values were normalized for virus amount as assessed by RT activity. HIV-1 was either produced by infected PBMCs cultured in collagen or suspension (infected cells), or sucrose-purified HIV-1 (virus) was directly embedded within media (suspension) or collagen for 24 h. Relative infectivity per µU RT is plotted relative to suspension, which is set to 100%. Mean+/−SD is indicated. Individual symbols indicate independent experiments. Wilcoxon matched pair test was used for statistical analysis
