Structural analysis of herpes simplex virus by optical super-resolution imaging

Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

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 1IGT 39 ). 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 .