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

Abstract

Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combination with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle averaging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

“…For applications in cell biology and molecular self-assembly reactions [7][8][9][10][11][12] that require imaging with high temporal resolution over many time points, structured illumination microscopy (SIM) can be well-suited as it is not dependent on the photophysical properties of a particular fluorescent probe. Despite this inherent advantage of SIM, up to now its use has been mainly confined to imaging fixed cells or slow moving processes.…”

Herpes Simplex Virus Capsid Localization to ESCRT-VPS4 Complexes in the Presence and Absence of the Large Tegument Protein UL36p

“…An essential component of the envelopment apparatus is the conserved multifunctional protein UL36p (VP1/2) (17)(18)(19)(20)(21). UL36p is the largest structural polypeptide encoded by the members of the Herpesviridae (22,23), and it forms the innermost layer (18,(24)(25)(26)(27)(28)(29)(30)(31)(32) of tegument, the complex protein scaffold between the capsid and envelope (8,16,33). UL36p attaches the capsid (18,31,32,34) to multiple outer tegument components (24-30, 35, 36) that in turn bind integral membrane envelope proteins (1,9,(37)(38)(39)(40) and the lipid envelope (41)(42)(43).…”

“…UL36p is the largest structural polypeptide encoded by the members of the Herpesviridae (22,23), and it forms the innermost layer (18,(24)(25)(26)(27)(28)(29)(30)(31)(32) of tegument, the complex protein scaffold between the capsid and envelope (8,16,33). UL36p attaches the capsid (18,31,32,34) to multiple outer tegument components (24-30, 35, 36) that in turn bind integral membrane envelope proteins (1,9,(37)(38)(39)(40) and the lipid envelope (41)(42)(43). One important function of UL36p is to recruit UL37p (19,20,25,29,44,45), a putative mimic of cellular multisubunit tethering complexes (28) that mediates capsid docking to organelles, including the trans-Golgi network (TGN) (27), perhaps via membrane anchors, such as the heterodimer gK/UL20p (46).…”

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids

“…Interestingly, capsids recruit pUL36 of residues 1 to 2894 that includes this pUL25 binding site during assembly but do not hold onto it during nuclear targeting upon cell entry (26). Superresolution fluorescence microscopy of extracellular HSV-1 virions localizes pUL36 in close proximity to the capsids, whereas pUL37 and VP16 are shifted toward the envelope consistent with pUL36's proposed structural linker function (53).…”