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.

Two-colour dSTORM resolves virus particles in infected cells. Next, we imaged virus particles within infected cells by using twocolour 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 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6980 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 cell 35 . 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( þ )/Tegument( þ )/Envelope( þ )) and others that were devoid of envelope (Capsid( þ )/Tegument( þ )/ Envelope( À )) (Fig. 1c) . The particles exhibiting all viral components (Capsid ( þ )/Tegument( þ )/Envelope( þ )) 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.