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.

For gD, the reduction in diameter from secondary to primary labelling is more pronounced than for the tegument proteins (B20 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 B20 nm on the diameter corresponds to B10 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.