Multi-colour dSTORM resolves protein layers in HSV-1

We used two-colour dSTORM imaging of purified virions to investigate the architecture of different protein layers within individual virus particles. The use of Alexa Fluor (AF) 647 is well established in localization microscopy, owing to its favourable photoswitching behaviour and its high photostability and quantum yield. We thus searched for a second fluorophore that could be reliably used in combination with AF647 for imaging virus particles and tested AF647/ATTO532, AF647/AF546 and AF647/AF568 in a range of photoswitching environments. All fluorophores were linked to secondary antibodies and imaging protocols were optimized for each fluorophore combination (Fig. 1a).

Figure 1: Multi-colour dSTORM analysis of purified and cellular HSV-1 particles. (a) Representative two-colour dSTORM images of purified virus particles obtained from various combinations of co-labelling, showing the relative position of protein layers within individual virus particles. Top panel shows labelling of the envelope and the tegument, whereas bottom panel presents labelling of two tegument proteins. (b) Four-colour imaging of HSV-1 in a fixed HFF-TERT cell. The nucleus is indicated by a dashed line. Cells were infected with VP26-mTurquoise/gM-EYFP recombinant virus for 12 h and then immunostained against gD (AF647) and VP1/2 (AF568), using secondary labelling. In the two insets (1, 2) showing zoom-ins, the two super-resolved images are overlaid (AF568 VP1/2 and AF647 gD, green and magenta, respectively) and the mTurquoise (mTQ) and EYFP channels are displayed. (c) Representative capsid-positive virus particles observed in cells showing the presence or absence of envelope. Scale bars, 100 nm (a,c), 5 μm (b), 1 μm (b1, b2). Full size image

As shown in the top row in Fig. 1a, for every fluorophore combination tested, two-colour dSTORM consistently demonstrated the envelope protein gD to be located at larger radial distance than the tegument protein VP16. The two tegument proteins VP16 and pUL37 showed a large overlap and similar radial distributions, suggesting that VP16 and pUL37 reside in the same region of the tegument layer (Fig. 1a, bottom row). The inner tegument protein VP1/2 and the outer tegument protein VP16 are thought to reside at different radial locations, with VP1/2 expected to lie closer to the capsid than VP16. However, by resolving and analysing individual virus particles, we could not consistently differentiate between outer and inner tegument, irrespective of dye combination used (Fig. 1a, bottom row, second and third panel).

Two-colour dSTORM resolves virus particles in infected cells

Next, we imaged virus particles within infected cells by using two-colour dSTORM. Here, to aid identification of the virus assembly state, we produced a fluorescently tagged recombinant HSV-1 that genetically encoded the small capsid protein VP26 fused to mTurquoise and the envelope protein gM fused to enhanced yellow fluorescent protein (EYFP). We were therefore able to locate individual capsids and discriminate between enveloped and non-enveloped particles within the infected cells. AF647 and AF568 were then used to label envelope protein gD and tegument protein VP1/2, respectively. The capsid protein VP26 (shown in blue in Fig. 1b) was primarily present in the nucleus (where capsids are assembled) and, less abundantly, in the cytoplasm where the punctate pattern indicates the presence of individual capsids. The envelope protein gM (shown in yellow) was mostly present in the perinuclear region of the cytoplasm and near the plasma membrane, as expected for a viral envelope glycoprotein that undergoes vesicle-based transport through the secretory and endocytic compartments of the cell35. dSTORM imaging of both AF647 and AF568 allowed the identification and characterization of structural elements of individual particles (tegument and envelope) directly in the infected cells and the mTurquoise fluorescence image was used to identify the particles that were capsid positive. Among the capsid-positive particles, two main types were observed: those containing both tegument and envelope (Capsid(+)/Tegument(+)/Envelope(+)) and others that were devoid of envelope (Capsid(+)/Tegument(+)/Envelope(−)) (Fig. 1c). The particles exhibiting all viral components (Capsid (+)/Tegument(+)/Envelope(+)) displayed structures that were consistent with those observed in purified viruses (Fig. 1a). In particular, VP1/2 (tegument) consistently appeared to be contained within the gD layer (envelope). We observed no clear visual difference in the distribution of VP1/2 between virus particles that were envelope positive and envelope negative.

These data demonstrate that the use of two-colour dSTORM in combination with two-colour wide-field fluorescence (using genetically expressed mTurquoise-VP26 and gM-EYFP) provides super-resolution information about individual virions at different assembly steps.

However, we noted that the mean localization precision (defined further on as 1 s.d.) achieved for the two-colour dSTORM images obtained both in cells and in purified viruses was of the order of 10–15 nm, irrespective of the fluorophore and the antibody used. We also observed that the fraction of localization with high-localization precision (5–10 nm) was much greater for AF647 than AF568, AF546 or ATTO532. This observation is in agreement with AF647 being one of the brightest fluorophore commonly used for dSTORM.

Particle averaging and model-based analysis of HSV-1

Nanometre-scale structural analysis by SMLM is a developing field. However, it requires high spatial resolution and high labelling specificity, and typically involves complex data analysis. This complexity is notably due to the need for assessing effects of the localization error and the linker size between the protein of interest and the fluorescent label36,37. Here we present a structural analysis method taking these effects into account and apply it for the precise determination of the distribution of important tegument and envelope proteins in HSV-1.

We acquired single-colour dSTORM images of purified viruses labelled with AF647. Here, a recombinant virus expressing mTurquoise-VP26 was used. We imaged a large number (>50) of particles and data sets were analysed as shown in Fig. 2a (see also Supplementary Notes 1–3 and Supplementary Figs 1–3). We ensured that only fully assembled virus particles, for example, containing capsid, tegument and envelope, were used for the analysis. For this, capsid-positive particles were selected by creating a mask from the mTurquoise-VP26 fluorescence image. A second mask was also created from an additional wide-field fluorescence image using AF568 as described in the Methods section.

Figure 2: Virus particle alignment and model-based analysis. (a) Flowchart representing the main data analysis steps. The image analysis procedure was performed using a custom-written MATLAB routine. Briefly, the dSTORM image was masked using the mTurquoise and AF568 fluorescence image, and the circular Hough (cHough) transform was applied to detect the particles and determine their centres. The radial distribution of localization is obtained from the aligned localizations and then fitted to the MCV model. (b) MCV model. The effect of the labelling linker, the imaging (projection) and the localization error are taken into account. The ⊗ symbol represents the convolution operation (for more details, see Supplementary Notes 1–3). Full size image

Images of individual virus particles (as those shown in Figs 2a and 3) were typically reconstructed from 100 to 300 localizations, with a typical localization precision of 5–10 nm (1 s.d. as described by Mortensen et al.38, see Supplementary Note 3). By aligning the obtained particles, the localizations can be combined to reconstruct a high-density super-resolution image of a specific protein layer. Furthermore, with the large number of localizations obtained in the aligned data set, the radial distribution of the aligned localizations can be accurately retrieved and analysed to determine the position of this individual layer. For this, the radial distribution of localizations was fitted to a Monte-Carlo model of virus localization data set (Monte-Carlo-based virus (MCV) model, presented in Fig. 2b and Supplementary Note 2), developed assuming spherically symmetric virus particles. In this model, each viral protein is assumed to lie within a spherical shell described by two parameters: the shell diameter and the shell thickness. The model takes both the linker size and the localization error into account. The shell diameter and thickness are the structural parameters of interest and are both obtained by fitting, whereas the linker size and localization precision are first estimated and then fixed during the analysis (Supplementary Note 3). The shell diameter represents the average radial position of the protein of interest in a population of virus particles. On the other hand, the shell thickness obtained by our method is a result of several contributions: the actual thickness of the protein layer (the thickness of the shell shown in Fig. 2b), the variability of the diameter from particle to particle in a population of virus particles and any deviation from the spherical symmetry.

Figure 3: Aligned particle images and results from the model-based analysis. Virus images obtained from aligned particles (aligned) and representative individual particles (individual). In the table, results from the model-based analysis are shown. The total number of localizations and number of particles are also indicated. The errors shown in the table are the 95% confidence interval obtained from the fit. n.d., not determinable. The radial distribution and the optimal fit are shown in Supplementary Fig. 3. Scale bar, 100 nm. Full size image

First, we investigated the effect of the fixed parameters (linker size and localization precision) on the fitted parameters. The estimation of the shell diameter was found to be very robust with respect to the fixed parameters and errors of 50% in linker size or localization error estimates only introduced a bias of <5%. However, the shell thickness is more strongly affected by such errors. An overestimation of either linker length or localization precision leads to an underestimation of the shell thickness (and vice versa). Details can be found in Supplementary Note 4 and Supplementary Fig. 4.

The analysis presented here allows the quantification of both diameter and thickness of individual protein layers from localization data sets and provides much greater precision than single-particle imaging.

Direct labelling of viral proteins

To assess the effect of linker size, we compared three different antibody-based labelling methods: indirect immunochemistry (successive primary and secondary antibody labelling, termed secondary labelling), direct dye-conjugated primary antibody (primary labelling) and direct dye-conjugated Fab fragments (Fab labelling), all conjugated with AF647. We estimated the corresponding linker sizes as 20, 10 and 5 nm, respectively, based on the structure of the IgG (PDB 1IGT39). The analysis was performed for the envelope protein gD and the three tegument proteins VP16, pUL37 and VP1/2.

In general, Fab fragments are preferable for super-resolution microscopy due to their small size. However, the specificity of the Fab fragments we produced was often compromised and efficient labelling was only achieved using the anti-gD Fab fragment. This is likely to be due to antibody structure and/or dye conjugation within the antigen-binding region, leading to lower affinity or avidity interactions between the specific epitopes. Therefore, only results from anti-gD Fab fragment are shown. Figure 3 shows the image of the virus obtained from particle alignment as well as representative single-particle images obtained from the raw dSTORM images for each protein and labelling combination. The total number of localizations, the number of particles and the results from the MCV model fits are stated in the table. Radial distributions and the optimal fits for all data sets are shown in Supplementary Fig. 3.

Irrespective of the labelling approach, the envelope protein gD exhibited the largest diameter, VP16 and pUL37 showed a diameter consistent with their tegument location (between capsid and envelope), whereas the inner tegument protein VP1/2 appeared very close to the capsid (from the known capsid diameter of 125 nm5,6,40).

The reduction in the number of localizations that was observed when comparing the secondary with primary and Fab labelling strategies can be attributed to the reduction of the average number of fluorescent labels per antibody, from 3 to 4 on commercial secondary antibody to 1.0–1.5 for in-house labelled primary antibodies and Fab fragments. In addition, multiple secondary antibodies may bind to a single primary antibody. This would also contribute to the greater number of localizations observed with secondary labelling.

As our analysis takes into account the linker size, different labelling strategies should lead to the same results. Any differences can then be interpreted as deviations from the model (in particular, non-spherical or non-random distribution) or direct effect of the labelling on the particles (labelling affecting the particle size, for instance). In fact, we observe that for VP16, pUL37 and VP1/2, changing the labelling from secondary to primary resulted in a small reduction of the diameter (by 5–10 nm). This small reduction may be a result of the heavy loading of the tegument with large labels that occurs with secondary labelling, leading to an observed swelling of the particle. The compact and rigid structure of the capsid may also contribute towards pushing the labels outwards rather than inwards.

For gD, the reduction in diameter from secondary to primary labelling is more pronounced than for the tegument proteins (~20 nm as opposed to 5–10 nm). In this case, the difference can be explained by a deviation between the envelope protein structure and the model. Instead of being randomly distributed around the average radial position of the protein layer, the labels may have a tendency to stretch outwards, away from the centre of the particle, and therefore smaller labels admit a smaller bias. This results in an overestimation of the protein diameter due to the linker size, which is then more pronounced in the case of the secondary labelling. An additional ~20 nm on the diameter corresponds to ~10 nm on the radius, which is in agreement with the size of a single antibody. No significant reduction in the diameter of the protein layer was observed between the primary and the Fab labelling, suggesting that the precision of our measurements is not limited by the size of the labelling once the distance between the protein of interest and the fluorescent label is of the order of or below the size of a single complete antibody.

The measurements of thickness obtained for gD are consistent across the three different labelling strategies. Similarly, the thicknesses recovered by primary and secondary labelling for VP1/2 are in good agreement with each other. We note that for VP16 and pUL37, the use of secondary labelling did not allow the estimation of the thickness parameter. This highlights that the use of primary labelling allows the measurement of smaller thickness that secondary labelling cannot reliably determine. A thorough analysis of the effect of the linker size (Supplementary Note 5) showed that the shell thickness may appear non-determinable if the estimation of the linker size is incorrect. This analysis also demonstrated that the linker size for the secondary labelling is more variable (between 15 and 25 nm for this data set depending, on the protein labelled) than for primary labelling. This variability in linker size using the same labelling approach is very likely to introduce bias or non-determinable thickness when the linker cannot be estimated precisely. On the other hand, a linker size of 10 nm for primary labelling provided reliable and consistent results.

As the secondary labelling is more subject to biases for model-based analyses, we conclude that the primary labelling appears the most robust method to measure the diameter and thickness of a discrete protein layer and is therefore the most appropriate approach for dSTORM structural analysis.

Modelling HSV-1 protein layer architecture

From the model-based analysis using primary labelling, the diameter of different protein layers could be obtained with high precision (1–2 nm error, 95% confidence interval). As shown in Fig. 4a, the diameter obtained decreased from envelope (171.4±2.4 nm) to the tegument proteins VP16 (153.8±1.4 nm), pUL37 (150.6±1.6 nm) and VP1/2 (121.8±1.8 nm). Moreover, the distance between the capsid and the envelope (23±4 nm) can be interpreted as a measurement of the average tegument thickness.

Figure 4: Model of protein distribution inside the tegument of HSV-1. (a) Average diameter of protein layers, error bars are 95% confidence interval obtained from the fit. (b) HSV-1 protein architecture model. This model was built from the diameters obtained using primary labelling and biochemical evidences of interaction sites and antibody-binding sites. *The diameter shown for the capsid was obtained from published EM data5. Full size image

The diameter obtained here for VP16 is in good agreement with the observation that it interacts with a number of envelope-anchored proteins41,42 and therefore is expected to localize close to the envelope. For VP1/2, it has been shown that the C-terminus of VP1/2 interacts with the minor capsid protein pUL25 (ref. 43), but the structure and the spatial arrangement of the remaining part of this large protein remains elusive. Here we were able to show that the binding site for the VP1/2-specific antibody used here (located between the amino acids 1564 and 1876) resides very close to the edge of the capsid. Furthermore, despite the common view of pUL37 as an inner tegument protein (notably because it interacts with VP1/2 (ref. 12)), the data we obtained here provide evidence that pUL37 is located at a similar position in the tegument to VP16 and, hence, closer to the outer part of the virion than previously thought.

From our measurements, we propose a model for HSV-1 tegument architecture (Fig. 4b). In addition, this model includes the current understanding of the interaction and antibody-binding sites of the considered proteins in the tegument. Indeed, we have previously shown that VP16 binds to VP1/2 within the first 361 amino acids of VP1/2 (ref. 14). Moreover, the binding site of VP1/2 to pUL37 has been localized within the C-terminal region of pUL37 (ref. 11). The diameter measured for pUL37 (153.8 nm) is in good agreement with published data showing that the interaction site of VP1/2 with pUL37 is close to the VP16 interaction domain, within the amino terminus of VP1/2 (ref. 44), potentially placing both VP16 and pUL37 in a similar location relative to VP1/2.

Although interpretation is not as straightforward as for the shell diameter data, the shell thickness also provides structural information. For instance, as the gD protein layer is part of the membrane structure (with no intrinsic thickness), the large thickness obtained for gD (40 nm) indicates a large variability of the envelope diameter from particle to particle (supporting the large range of particle sizes found by EM) and/or a potential deviation from the spherical symmetry, underlying the plasticity of the envelope. On the other hand, the thickness of VP16 and pUL37 appear smaller than that of gD, which is consistent with the notion that there is much less plasticity in the virus tegument compared with the envelope layer. At the moment, we do not have a clear explanation as to the observed differences in the thickness for VP1/2 and pUL37. Reasons could include differential labelling efficiencies for the two proteins and dense packing of fluorophores on the small shell of VP1/2, increasing localization artefacts. An underestimation of the alignment error would also lead to an overestimation of the shell thickness (see Supplementary Note 4). Finally, there may be differential ‘plasticity’ of the two protein layers, leading to a real geometric distortion.

Spatial offset of the capsid

Cryo-ET studies indicated that HSV-1 particles are not symmetrical4,45. These studies demonstrated that the capsid does not lie in the centre of the particle. This spatial offset may be explained by the structure and function of the portal vertex, which is thought to form a connection between the capsid and the envelope45. A spatial offset between the centre of the capsid and the outer protein layer of ~15 nm was measured by ET4.

Here we developed a method to estimate the average distance between the centre of the capsid and the centre of the outer protein layer (tegument or envelope), as detailed in Supplementary Note 6. Briefly, the displacement between the centre of the considered layer (tegument or envelope, determined by a fitted circle) and the centre of the capsid (determined by localization of the centroid of the mTurquoise-VP26 fluorescence) was calculated for a large number of particles and the data were analysed with a further model-based analysis. This approach is related in nature to that of a recent investigation of sub-virion protein distribution in the related herpesvirus pseudorabies virus46. In the latter work, the displacement between the centroids of the wide-field fluorescence patterns from dually labelled virions was determined with high precision. The results of our analysis are shown in Supplementary Fig. 5b. After correction for the residual chromatic random error, the offset was estimated as 12, 7 and 24 nm for gD, VP16 and pUL37, respectively. The order of magnitude of this offset is in good agreement with the value determined by ET4, although greater than expected for pUL37.