Quantitative Correlation between Infectivity and Gp120 Density on HIV-1 Virions Revealed by Optical Trapping Virometry

Abstract

The envelope glycoprotein (Env) gp120/gp41 is required for HIV-1 infection of host cells. Although in general it has been perceived that more Env gives rise to higher infectivity, the precise quantitative dependence of HIV-1 virion infectivity on Env density has remained unknown. Here we have developed a method to examine this dependence. This method involves 1) production of a set of single-cycle HIV-1 virions with varied density of Env on their surface, 2) site-specific labeling of Env-specific antibody Fab with a fluorophore at high efficiency, and 3) optical trapping virometry to measure the number of gp120 molecules on individual HIV-1 virions. The resulting gp120 density per virion is then correlated with the infectivity of the virions measured in cell culture. In the presence of DEAE-dextran, the polycation known to enhance HIV-1 infectivity in cell culture, virion infectivity follows gp120 density as a sigmoidal dependence and reaches an apparent plateau. This quantitative dependence can be described by a Hill equation, with a Hill coefficient of 2.4 ± 0.6. In contrast, in the absence of DEAE-dextran, virion infectivity increases monotonically with gp120 density and no saturation is observed under the experimental conditions. These results provide the first quantitative evidence that Env trimers cooperate on the virion surface to mediate productive infection by HIV-1. Moreover, as a result of the low number of Env trimers on individual virions, the number of additional Env trimers per virion that is required for the optimal infectivity will depend on the inclusion of facilitating agents during infection.

Keywords: glycoprotein; human immunodeficiency virus (HIV); infection; optical tweezers; protein cross-linking; single particle analysis; single-molecule biophysics.

FIGURE 1.

Specific labeling of VRC01 Fab by Alexa 594-maleimide. a, schematic of a full-length IgG. b, site-specific labeling strategy for VRC01 Fab. c, silver staining to assess the purity of Alexa 594-labeled VRC01 Fab using a 4–15% gradient polyacrylamide gel. The 100% corresponds to 3 μg of purified protein. d, neutralization of NL4-3 virions by purified VRC01 Fab.

FIGURE 2.

VRC01 antibody induces negligible shedding of NL4-3 gp120. a, Western blotting analysis of gp120 and gp160 proteins in the culture supernatant upon incubation with VRC01 IgG at various concentrations. b, quantitation of the gp120 band intensity from panel a as a function of VRC01 IgG concentration. The positive control for this assay, demonstrating gp120 shedding in response to addition of various concentrations of CD4 under identical cell and incubation conditions, was published previously (10).

FIGURE 3.

TPF intensity of single Alexa-594 molecules on HIV-1 virions. a–c, representative TPF time trajectories from Alexa 594 molecules associated with individual HIV-1 virions (gray traces). The step-finding algorithm that we developed previously (19) was used to identify steps from these real time trajectories, which are shown as red lines. d, the histogram of the individual photobleaching step sizes for Alexa 594 molecules identified from the step-finding algorithm (n = 303), which could be described by the sum of two Gaussians (red curve).

FIGURE 4.

Histograms for the number (#) of gp120 molecules per virion determined from OTV. The histograms, with gray, black, blue, orange, purple, red, and green curves are for virions from 0 (n = 80), 0.01 (n = 79), 0.1 (n = 105), 0.2 (n = 119), 1.0 (n = 129), 2.0 (n = 85) and 4.0 μg of pEnv (n = 69), respectively.

FIGURE 5.

Relationship between HIV-1 virion infectivity and the average number of gp120 molecules per virion in the presence (a) or absence (b) of DEAE-dextran. The symbols are experimental data, where the error bars for infectivity are standard deviations derived from three independent repeats of the same experiment, and the error bars for the average number of gp120 molecules per virion are mean ± S.E. derived from single-particle data. The adjusted R-square values for the fittings are 0.9975 and 0.8293, respectively. The dashed curve in panel b shows a sigmoid function with large uncertainties in the fitted parameters.

FIGURE 6.

Relationship between HIV-1 virion infectivity and the average number of gp120 molecules per Env⁺ virion in the presence (a) or absence (b) of DEAE-dextran. The error bars for infectivity are standard deviations derived from three independent repeats of the same experiment, and the error bars for the average number of gp120 molecules per Env⁺ virion are mean ± S.E. derived from single-particle data. The data points for virions prepared with pEnv = 0.01 and 0.1 μg are labeled.