Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture

Abstract

The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection.

Fig. 1.

Acquisition of fluorescent proteins in to viral particles. (A and B) Fluorescence micrographs of a single diffraction-limited PRV particle expressing gD-GFP (A) and RFP-pUL35 (B). (C) Example of a red-green displacement measurement using simulated data: 2D Gaussian curves are shaded red and green, vertical dashed lines are the centroids, and the double-headed arrow is the measured displacement. (D–O) Micrographs of dual-fluorescent PRV particles. (D–I) mRFP1-pUL35 capsids. (J–O) corresponding GFP in matched fields. Where L-particles are abundant, arrowheads represent the position of H-particles. All exposures were 1.5 s. Images of the same color have equivalent intensity scaling. (Scale bar, 10 µm.)

Fig. 2.

Capsid fluorescence properties of recombinant H-particles. (A) Representative frequency distribution of red capsid fluorescence. Red fluorescence intensity (x axis) is expressed in arbitrary units. Nonlinear regression was used to fit a normal distribution to the histogram. For all strains, the goodness of fit (R²) of the data to a normal distribution was greater than 0.95. (B) The average red fluorescence particle intensity for each recombinant virus. The x axis indicates intensity values expressed as arbitrary fluorescence units. Error bars represent the SEM for n ≥ 3 independent experiments. *P < 0.05, as determined by one-way ANOVA and a post hoc Tukey test.

Fig. 3.

Incorporation of GFP-fusion proteins into viral particles. (A) Viral particles were scored positive for GFP-RFP colocalization if GFP was detectable in RFP⁺ particles after background subtraction. (B) Estimated copy number of GFP-fused proteins in PRV virions. Copy number was determined by dividing the mean green fluorescence of triplicate experiments by the mean fluorescence of pUL25/GFP and multiplied by 70.5 (see text). For pUL47, the exposure time was reduced from 1.5 s to 0.5 s, and the diagonal fill represents the normalized value based on linear extrapolation. (C) CV of GFP fluorescence for each strain. CV was obtained for each sample by dividing the SD of each sample by its mean. Error bars represent SEM for n ≥ 3 independent experiments.

Fig. 4.

Comparison of L- and H-particle protein incorporation. The x axis indicates intensity values expressed as arbitrary fluorescence units. H-particle fluorescence (filled circles) was fit to a normal distribution (solid line), and L-particle fluorescence (empty circles) was fit to a decaying exponential distribution (dashed line). All L-particle decaying exponentials had a goodness of fit R² > 0.98. Normal distributions fit H-particle data with R² > 0.95, with the exception pUS3-GFP (R² = 0.89), pUL46-GFP (R² = 0.88), and UL47-GFP (R² = 0.92), pUL48-GFP (R² = 0.92), and gD-GFP (R² = 0.93).

Fig. 5.

Subvirion protein distributions. (A) Models of predicted displacement values for green fluorescent proteins (blue: postulated distribution; green: centroid of fluorescence emission) from the capsid (red: centroid of capsid fluorescence). Models were derived by determining the centroids of icosahedra (Top), and spheres, which were solid (Middle) or hollow (Bottom). A capsid eccentricity of 15 nm was modeled based on measurements of HSV-1 by Grünewald et al. (8). (B) Displacements of green fluorescence from the red fluorescent capsid. As illustrated in Figs. 1C and 2D, Gaussian curves were used to determine the point source of Airy disks in red and green channels. The bar graph shows the average displacement of the green fluorescence centroid from the red fluorescence centroid. Blue shaded regions indicate likely locations of proteins within the virion based on the models in A. The center of the shaded regions represents the mean expected value, and width represents 1 SD to either side of the mean. The five bars shaded in light gray are not significantly different from one another (see text). Error bars represent the SEM for n ≥ 3 independent experiments.