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 of 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 the 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.IMPORTANCEAs the prototypical herpesvirus, herpes simplex virus 1 (HSV-1) exhibits a global seroprevalence of 67% and approaching 90% in some localities. Herpesvirus infections can cause devastating cancers and birth defects, with HSV-1 infections leading to cold sores among the general population worldwide and blindness in developing nations. Here, we present atomic structures of the capsids sorted out from the nuclear isolates of HSV-1 infected cells, including the previously recognized A-, B-, and C-capsids, as well as the newly identified D-capsid. The structures show the details of protein-protein and protein-DNA interactions within each capsid type and the positional and interactional variability of the viral DNA-translocating portal vertex among these capsids. Importantly, our findings suggest that A-capsids are products of failed dsDNA packaging and D-capsids of failed genome retention. Together, the high-resolution 3D structures clarify the processes of genome packaging, maintenance, and ejection during capsid assembly, which are conserved across all herpesviruses.

Keywords: HSV-1; cryoEM; human herpesviruses; procapsid; virus assembly.

Fig 1

Asymmetric reconstructions and comparisons of HSV-1 A-, B-, C-, D-, and virion nucleocapsids. (A–E) asymmetric reconstructions of A-, B-, C-, D-, and virion (EMDB: EMD-9864) capsids showing empty (A and D), scaffold-containing (B), and genome-containing (C and E) capsid shells. The color bar on the right indicates radial distance from the capsid center in Å. (F–J) Asymmetric reconstructions of the portal vertices from A-, B-, C-, D-, and virion (EMDB: EMD-9860, EMD-9862) capsids in A–E. The orange dashed line in G and H labeled with “65 Å” indicates the difference in turret helix height across capsid types. Lower orange dashed line labeled with “14 Å” indicates the difference in portal basket height across capsid types. *Scaffolding density (lime) in F and G are not necessarily equivalent, as the density in A-capsids is probably a fragmentary remnant after proteolytic cleavage. SCP, small capsid protein; MCP, major capsid protein; Tri1, triplex 1 protein; Tri2, triplex 2 protein; CVSC, capsid vertex specific component. (K) Comparison of genome organization in D-, C-, and virion portal vertices, showing consistent arrangement in virions, C-capsids, and some D-capsids, but symmetric, round density in another class of D-capsids. (L) Graph of prevalence of empty capsids in our study of HSV-1, as well as nuclear thin-section versus purified samples for HCMV using data from reference .

Fig 2

High-resolution structure of the portal basket. (A) From left to right: full portal dodecamer including turret helices colored by domain, resolved portions of a portal monomer, and examples of model fit to cryoEM density in the clip (green), turret (cyan), and wing (blue green) domains. (B) cryoEM density of the portal vertex showing orientation of top (red) view in panel C and bottom (blue) view in panels D and E. (C) Top view of the portal basket showing assignment of portal anchors (smooth outer blobs) to portal subunits, with subunits missing an anchor labeled in red. Hexagons indicate fivefold locations of hexons relative to the portal. (D) Bottom view of MCP (lavender) and Tri1 (purple) fragments near the portal anchors (dodger blue) with important domains of MCP (PDB: 6ODM) and Tri1 labeled. (E) Bottom view of MCP and Tri1 fragments near portal anchors colored by hydrophobicity. Bottom left and top black outlines indicate the views in panels F and G, respectively. (F and G) Views of the lone helix (left) and helix pair (right) of the portal anchors (blue outlines) overlaid with hydrophobic surface view of their binding pockets, with hydrophobic residues shown.

Fig 3

Portal basket flexibility and positional variability within each capsid type for HSV-1 A-, B-, C-, and D-capsids. (A–D) Portal basket models fit into alternative conformations found via 3D flex analysis for each of the four capsid types, with the extreme edges of their conformational ranges represented as salmon and aqua structures. Multiple separate analyses show vertical translation (left), rotation (center), and a swaying motion of the top half of the basket (right).

Fig 4

The scaffolding protein interacts with the portal dodecamer. (A–E) Ribbon diagram of modeled residues in the portal dodecamer for A-, B-, C-, D-, and virion capsids, including basket (salmon), turret helices (cyan), and where applicable, the associated genome (orange) and scaffolding protein loops (lime). (F–J) Zoom-ins of the ribbon model and density of the portal’s scaffold binding pocket for the five capsid types in (A–E), showing the presence or absence of scaffold density (lime). (K) Residues 449–455 of the scaffolding protein fit into a density interacting with the portal. (L) Interatomic distances between atoms of scaffolding and portal at their interaction site, highlighting hydrophobic residues. (M) Scaffolding loop shown with a hydrophobicity map of the portal, showing hydrophobic spots near Y451 and P452. (N) Sequence alignment of portal and scaffolding proteins across a range of herpesviruses, demonstrating sequence conservation of residues important for portal-scaffold interactions.

Fig 5

Global arrangement of the scaffold in whole B-capsids. (A) Cut view of whole B-capsid reconstruction showing large internal scaffold densities (top left), with Tri1 and Tri2 labeled in purple and red, respectively. (B) Unwrapped view of the capsid floor showing global scaffold arrangement, with penton locations marked with black-outlined white pentagons. (C) Scaffold densities at portal (blue dashed outline) and antipodal (green dashed outline) poles. (D) Comparison of the portal basket in HSV-1 procapsids from Buch et al. (44) (EMDB: EMD-22379) with our B-capsid reconstruction, showing 4.5 nm difference in position.

Fig 6

Proposed model of herpesvirus maturation and assembly. Virion capsids release the viral genome into the host cell nucleoplasm, where the genome is transcribed and translated to form viral proteins. These proteins are then imported into the nucleus and associate with a free portal complex via scaffold-mediated interactions, initiating assembly of the procapsid. If angularization occurs prematurely, B-capsids are formed. If angularization and scaffold digestion are completed but not genome packaging, A-capsids are formed. Successfully packaged capsids, or C-capsids, may either undergo further maturation into virions or fail to retain genome, resulting in the formation of D-capsids.