Structure of a new capsid form and comparison with A-, B- and C-capsids clarify herpesvirus assembly.

Abstract

Three capsid types have been recognized from the nuclei of herpesvirus-infected cells: empty A-capsids, scaffolding-containing B-capsids, and DNA-filled C-capsids. Despite progress in determining atomic structures of these capsids and extracellular virions in recent years, debate persists concerning the origins and temporal relationships among these capsids during capsid assembly and genome packaging. Here, we have imaged over 300,000 capsids of herpes simplex virus type 1 by cryogenic electron microscopy (cryoEM) and exhaustively classified them to characterize the structural heterogeneity of the DNA-translocating portal complex and their functional states. The resultant atomic structures reveal not only the expected A-, B-, and C-capsids, but also capsids with portal vertices similar to C-capsids but no resolvable genome in the capsid lumen, which we named D-capsids. The dodecameric dsDNA-translocating portal complex varies across these capsid types in their radial positions in icosahedral capsids and exhibits structural dynamics within each capsid type. In D-capsids, terminal DNA density exists in multiple conformations including one reminiscent to that in C-capsids, suggesting D-capsids are products of failed DNA retention. This interpretation is supported by varying amounts of DNA outside individual D-capsids and by correlation of capsid counts observed in situ of infected cell nuclei and those after purification. Additionally, an "anchoring" segment of the scaffold protein is resolved interacting with the portal baskets of A- and B-capsids but not D- and C-capsids. Taken together, our data indicate that A-capsids arise from failed DNA packaging and D-capsids from failed genome retention, clarifying the origins of empty capsids in herpesvirus assembly.