Fig 1.
gSTED reveals various features of glycoprotein organization at the surface of viral particles.
(A) Purified WT 17+ virions were attached onto a glass coverslip and labeled with mAb LP11 against gH/gL and an Oregon green 488-labeled secondary antibody. Images were acquired using the diffraction-limited confocal mode or with a gated STED set-up (gSTED). Images were further processed for noise-filtering using Huygens software algorithms. Scale bars: 500 nm. (B) Different patterns of glycoprotein organization were observed in the gSTED mode, which were not resolvable in the confocal mode. From top to bottom these were: single, double, and multiple spots, and rings. Three of the patterns correspond to particles boxed in (A). The single spot image is of a particle labeled with gC-specific mAb IC8 and is included here for illustrative purposes. Each different pattern is shown in the confocal and in the gSTED mode with corresponding noise-filtered images. Arrows delineate the line (500 nm long) used for profile analysis of normalized intensity. The blue line and the orange line in the graph correspond to the intensity profiles observed in the confocal and in the gSTED modes respectively. Analysis was carried out on raw, unprocessed images. Scale bars: 100 nm. (C) Schematic illustration as a 2D projection of the different patterns described in (B). The number of proteins per cluster (one or more) is unknown.
Fig 2.
Free and cell-bound particles may display different glycoprotein patterns.
Particles bound to cells (+) or attached to coverslips (-),were imaged using gSTED and the localizations of glycoproteins gB, gH/gL, gD and gC were assessed by using a monoclonal (mAb) or a polyclonal antibody (pAb) recognizing each glycoprotein. Localization was categorized as single (blue), double (green) or multiple spots (yellow) or rings (red), as described in Fig 1. (A) Cells used for attachment were HFFF cells. Particles were attached to glass coverslips at room temperature whereas particles were bound to cells at 4°C to prevent membrane fusion. No IC8-positive signal (gC) could be detected on cell-bound particles. (B) Cells used for attachment were HeLa cells. Particles were attached to glass coverslips or bound to cells at 4°C. Localization of gC was not analyzed in this experiment. A Pearson’s chi-squared test was used to determine whether the profile of distribution of one glycoprotein was statistically different between free and cell-bound virions. The p-value indicates the likelihood of a correlation, therefore a p-value > 0.05 was considered as indicating a statistically significant difference between the two sets. ns: p<0.05, **: p>0.1, ***: p > 0.5. n: number of analyzed particles per condition. ND: not determined.
Fig 3.
The diameter of labeled rings may differ between glycoproteins and between free and cell-bound viral particles.
The diameters of 2,467 rings observed on raw, unmodified images obtained in gSTED mode were measured and compared according to the type of glycoprotein labeled and whether the particles were bound to cells or not. (A) Purified virions were attached to glass coverslips at room-temperature (Free) or bound to HFFF cells at 4°C. (B) Purified virions were attached to glass coverslips at 4°C (Free) or bound to HeLa cells at 4°C. Statistical differences were measured using an unpaired student’s t-test after the Gaussian distribution of values was verified using a Shapiro-Wilk normality test. ns: p >0.01, ***: p<0.0001 (B). n: number of analyzed particles per condition.
Fig 4.
Distribution of glycoproteins on cell-free on cell-bound viral particles.
Representative noise-filtered gSTED images of glycoprotein distribution on the surface of free or HFFF-bound viral particles for all available antibodies (see S3 Fig). The intensity profile of one particle per field along a 500 nm-long line delineated by two arrows is shown above each picture. Blue line: confocal profile; orange line: gSTED profile. The intensity profile was determined from the unmodified raw images. The peak-to-peak distance is indicated for each gSTED profile. No specific signal could be observed with the IC8 antibody on cell-bound particles. Scale bars: 500 nm.
Fig 5.
Relative positions of glycoproteins on free virus particles as revealed by dual-color gSTED.
(A) Purified free particles were double-labeled with twelve different pairs of antibodies. In each case, the percentage of particles labeled with mAb only is shown in green, the percentage of particles labeled with pAb only is shown in red and the percentage of double-labeled particles is shown in yellow. n: number of analyzed particles per condition. (B) For every pair of antibodies, representative noise-filtered gSTED pictures of cell-free particles are shown. The images on the left show more commonly observed patterns while those on the right are less commonly observed. mAb staining is pseudo-colored in green and pAb staining is pseudo-colored in magenta. All pictures are 700 x 700 nm.
Fig 6.
Positions of glycoproteins relative to gD on HeLa-bound virus particles as revealed by dual-color gSTED.
(A) Viral particles were attached to glass coverslips or bound to HeLa cells at 4°C and labeled with five different pairs of antibodies. In each case, the percentage of particles labeled with mAb only is shown in green, the percentage of particles labeled with pAb only is shown in red and the percentage of double-labeled particles is shown in yellow. (B) For every pair of antibodies, two different sets of representative noise-filtered gSTED pictures of HeLa-bound particles are shown. mAb staining is pseudo-colored in green and pAb staining is pseudo-colored in magenta. All pictures are 700 x 700 nm. (C) The distribution of two glycoproteins per condition was determined on free particles (-) or on particles bound to HeLa cells (+) and the different combinations were categorized into eight different profiles. The profiles used here illustrate combinations such as “ring-ring” (red bars), “ring-spot(s)” (orange bar), “spot(s)-ring” (yellow bar), “spot(s)-spot(s)” (green bar), “ring-no signal” (blue bar), “no signal-ring” (pink bar), “spot(s)-no signal” (purple bar) and “no signal-spot(s)” (grey bar) for “mAb-pAb” signals (colored in green and pink respectively). Single spots are shown here for illustrative purposes but spots can also be multiple. A Pearson’s chi-squared test was used to determine whether the profile of distribution of one glycoprotein was statistically different between free and cell-bound virions. A low p-value indicates the likelihood of a correlation, therefore a p-value > 0.05 was considered as indicating a statistically significant difference between the two sets. ns: p<0.05, **: p>0.1, ***: p > 0.5. n: number of analyzed particles per condition.