Organization of capsid-associated tegument components in Kaposi's sarcoma-associated herpesvirus

Abstract

Capsid-associated tegument proteins have been identified in alpha- and betaherpesviruses to play an essential role in viral DNA packaging. Whether and how such tegument proteins exist in gammaherpesviruses have been mysteries. Here, we report a 6-Å-resolution cryo-electron microscopy (cryo-EM) structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gammaherpesvirus subfamily. The KSHV virion structure reveals, for the first time, how capsid-associated tegument proteins are organized in a gammaherpesvirus, with five tegument densities capping each penton vertex, a pattern highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region, and a β-sheet-rich triplex-binding region. Fitting of the crystal structure of the truncated HSV-1 UL25 protein (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 established that ORF19 constitutes the globular region with an N-terminal, 60-amino-acid-long helix extending into the stalk region. Matching secondary structural features resolved in the cryo-EM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alphaherpesvirus UL17. Despite the high level of tegument structural similarities between KSHV and alphaherpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alphaherpesviruses, binds each penton subunit, an observation that correlates with conformational differences in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a long-standing mystery surrounding the virion location and conformation of alphaherpesvirus UL25 protein.

Importance: Several capsid-associated tegument proteins have been identified in the alpha- and betaherpesvirus subfamilies of the Herpesviridae. These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gammaherpesviruses, the third herpesvirus subfamily, which contains members causing several human cancers. Here, by high-resolution cryo-EM, we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gammaherpesviruses, KSHV. The cryo-EM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alphaherpesvirus UL25 and UL17 homologs in KSHV, respectively) in the KSHV capsid-associated tegument cryo-EM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alphaherpesvirus UL25 protein inside the virion.

FIG 1

Cryo-EM and 3D reconstruction of KSHV virions. (A) Cryo-EM image of purified KSHV virion. (B) 3D reconstruction of the KSHV virion at 6-Å resolution. The structure is rainbow-colored radially. The left half of the capsid is rendered at a contour level (CL) of 2δ (δ is the standard deviation), while the right half is rendered at 0.5δ to show the presence of tegument densities. (C) One-quarter of a central slice of the KSHV virion reconstruction. The white arrow points to a capsid-associated tegument density surrounding the penton. The intensity of the tegument density is measured to be ∼30% of the peak intensity of the capsid shell. Lines 2, 3, and 5, icosahedral symmetry axes. (D and E) Zoomed-in views of capsid surface areas as denoted with dashed squares in panel B. The black arrow in panel D points to a tegument density around the capsid vertex. Black arrows in panel E point to tegument density close to the 2-fold axis. 5, 5-fold axis; P, periphery-hexon; Ta and Tc, triplexes; 2, 2-fold axis; E, edge hexon; C, central hexon; Tb and Te, triplexes. (F) KSHV MCP upper domain (MCPud) density map (transparent gray) fitted with the HSV-1 MCPud atomic model (red ribbon; PDB entry 1NO7) (37).

FIG 2

Comparison of capsid-associated tegument densities in KSHV and pseudorabies virus (PRV). (A and B) Densities around the capsid vertex in the 6-Å KSHV virion reconstruction (A) and 9-Å PRV C-capsid reconstruction (B) (rendered from EMDB-5650; published by Homa et al. [17]). In both structures, capsid proteins are rainbow colored radially and tegument densities are gray, and they are displayed at a lower contour level than that of the capsid because they are weaker than capsid densities. (C) Schematic representation of pentons and periphery hexons in KSHV and PRV. The PRV penton has a 30° clockwise rotation when its periphery hexons are aligned to those of KSHV. (D and E) Zoomed-in views of a single capsid-associated tegument density in KSHV and PRV. Their positions are denoted with dashed squares in panels A and B. Note the slightly different orientations of the tegument density relative to the penton MCP. In KSHV (D), a single copy of the HSV-1 UL25 atomic model (magenta ribbon; PDB entry 2F5U) (39) can be fitted into the tegument density, while in PRV (E) two copies (magenta and blue ribbons) can be accommodated. (F) Two copies of the HSV-1 UL25 atomic model (magenta and blue ribbons) fitted in the tegument density of the PRV virion reconstruction (light blue) (EMDB-5655; Homa et al. [17]). Note the good match between the overall shape of the density map and that of the model. (G and H) Side views of tegument density interacting with penton and triplexes Ta and Tc in KSHV (G) and PRV (H), respectively.

FIG 3

Identity of constituent proteins in KSHV capsid-associated tegument component. (A and B) Different views of the KSHV tegument density. For convenience of description, the density is divided into a penton-binding globular region, a stalk region, and a triplex-binding region. The density is colored by its putative identity (magenta, ORF19; gray, ORF32; blue, unidentified tegument protein). The green symbol in panel A roughly corresponds to the position of extra GFP density in the HSV-1 UL25 GFP-labeling study as described by Conway et al. (15). The green symbol in panel B roughly corresponds to the position of GFP label inserted at the HSV-1 UL17 C terminus as described by Toropova et al. (16). (C) Zoomed-in view of the penton-binding globular region. The density map is presented as semitransparent and from an orientation different from that shown in panels A and B to appreciate its satisfactory fitting with the HSV-1 UL25 atomic model (magenta ribbon). This evidence strongly supports our assignment of ORF19 (UL25 homolog) to this region. The black arrows point to several well-fitted alpha-helices. (D) In situ, full-length structure of ORF19. N′ and C′ denote protein amino and carboxyl termini, respectively. The green mark is the same as that in panel A. (E) Schematic model of ORF19. Secondary structure prediction suggests that the N-terminal segment (aa 1 to 124 in KSHV ORF19, corresponding to aa 1 to 133 in HSV-1 UL25) missing from the HSV-1 UL25 crystal structure (aa 134 to 577) has a long helix and a short β-strand. (F) Structure of ORF32 (UL17 homolog). The green mark is the same as that described for panel B. N′ and C′ denote protein amino and carboxyl termini, respectively. (G and H) Zoomed-in views of the two domains of ORF32 to show its β-sheet-rich structural feature (colored yellow). (I) Secondary structure prediction of the ORF32 sequence. Note the abundance of β-strands, correlating with structural features shown in panels G and H. (J) Structure of the three-helix bundle in the stalk region.

FIG 4

Schematic presentation of capsid-associated tegument component organizations in gammaherpesvirus (A) and alphaherpesvirus (B). The main differences are the following: (i) only one ORF19 molecule is present in each gammaherpesvirus tegument density, compared to two copies of UL25 in each alphaherpesvirus tegument density; (ii) ORF19 binds only one penton MCP in gammaherpesvirus, while each UL25 in alphaherpesvirus binds two penton MCPs due to a 30° clockwise rotation of the penton, denoted as described in the legend to Fig. 2C.