Fig 1.
The 3D reconstruction of MCMV icosahedral capsid.
(A) Radially colored surface representation of the 3D reconstruction of MCMV at ~5 Å resolution, viewed along a 3-fold axis. Only the icosahedral symmetric components, including the capsid and capsid-associated tegument complex (CATC), are visible because the pleomorphic envelope and tegument layer, as well as the unique portal complex, have been smeared due to averaging of tens of thousands of particles and imposition of icosahedral symmetry. The filled pentagon, triangle and oval mark a 5-fold, 3-fold, and 2-fold axis, respectively. (B) Zoom-in surface view of one facet of the icosahedral capsid with structural components colored as indicated. Density of triplex Tf region at the center is smeared due to imposition of 3-fold symmetry during icosahedral reconstruction. A slightly lower density threshold was used for the pentons and triplex Ta/Tf with their associated tegument protein pM32, such that their volumes are comparable to subunits elsewhere. (C-D) Schematic (C) and cryoEM density map (D) of one asymmetric unit (shaded) of the reconstruction with individual capsid protein subunits labeled, following the nomenclature used in HCMV [14]. Subunits of triplex Tf and its associated pM32 are shown in semi-transparency instead of solid colors as their orientations were not determined in the icosahedral reconstruction here, but inferred from sub-particle reconstruction after sub-particle 3D classification (see S3 Fig).
Fig 2.
Resolution assessment, sub-particle reconstructions and MCMV atomic models.
(A-D) The icosahedral reconstruction in Fig 1A is reshown (A) with one triangular facet in color and regions (referred to as “sub-particles”) surrounding a 5-fold, 3-fold, and 2-fold axis circled. A total of 575,784 5-fold sub-particles, 959,640 3-fold sub-particles, and 1,439,460 2-fold sub-particles were boxed out from original particle images and refined to yield improved resolutions for the 5-fold sub-particle (B), 3-fold sub-particle (C), and 2-fold sub-particle (D). The enlarged views from inside (right panels) of these sub-particle reconstructions show α-helices and β-strands with well-resolved side chain densities. (E) Gold-standard (0.143) Fourier shell correlation (FSC) curves of the sub-particle reconstructions indicating the resolutions of sub-particle reconstructions at the 5-fold (red), 3-fold (blue), and 2-fold (green) axes are 3.8 Å, 3.6 Å, and 3.8 Å, respectively. (F-G) Close-up views of cryoEM density map (gray mesh) of an α-helix (F) and a loop (G) in the MCP floor region, superposed with atomic models (color). (H) De novo atomic models of individual capsid (MCP, SCP, Tri1, Tri2A, and Tri2B) and tegument (pM32nt) proteins shown as rainbow-colored ribbons (blue at the N-terminus to red at the C-terminus).
Fig 3.
MCP structure and three types of capsid floor-defining MCP-MCP interactions.
(A) Domain organization in the hexon MCP. (B) The structure of the Johnson-fold domain shown in rainbow-colored ribbon with sub-domains labeled. (C-D) Comparison of the buttress domain in hexon (C) and penton (D) MCP. An elbow-like helix-turn-helix structure (magenta in C) in the buttress domain of hexon MCP is folded into a single long helix (magenta in D) in penton MCP. (E) Overview of MCPs from C, E, and P hexons. Local 3-fold and 2-fold axes are indicated by a triangle and an oval, respectively. Three major types of MCP-MCP interactions are boxed in cyan (type I), red (type II), and yellow (type III), respectively. (F) Details of the three types of MCP-MCP interactions.
Fig 4.
(A) SCP shown as a rainbow-colored ribbon in two orthogonal reviews. (B) SCP binding to the upper domain of hexon MCP, as seen from the side of two adjacent MCP subunits (left panel) and from the top of a P hexon (right panel). Only MCP upper domains are shown. (C) Pipe-and-plank depictions of a penton (left panel) and hexon (right panel) with SCP and MCP subunits shown in red and gray, respectively. (D-E) SCP (ribbons) bound to the MCP upper domain, shown either as surface (D) with the SCP-binding groove highlighted in pink, or as ribbons with the hydrophobicity properties of the structures around the SCP-binding groove in color (E). SCP-MCP interactions are mainly hydrophobic.
Fig 5.
Structure of triplex and functional significance of Tri1 N-anchor.
(A) Distribution of triplexes Ta, Tb, Tc, Td, and Te among penton and three types of hexons (C, E, and P). (B) Enlarged view of a triplex Td with three adjacent hexon MCP subunits labeled (C1, E5, and P3). (C-D) Details of the structures of triplex Td (C) and Tri1 (D). (E) Bottom view of (B) showing that triplex Td anchors to the capsid floor by the “V”-shaped Tri1 N-anchor. (F) Triplex Td viewed from inside of the capsid showing similar clamp and trunk domains, albeit rotated about 120° relative to each other. (G) Pipe-and-plank representations of Tri2 dimer in side (left panel) and top (right panel) views showing the helix bundle formed from Tri2A and Tri2B’s embracing arm domains. (H) Superposition of Tri2A and Tri2B showing nearly identical clamp and trunk domains, but different embracing arms.
Fig 6.
Comparison of capsid-binding patterns of CATC in MCMV and HCMV.
(A) A triangular facet of MCMV icosahedral reconstruction. Except for triplex Tc (highlighted in yellow), two pM32nt subunits form a “Λ”-shaped density (cyan and orange-red) acts like a stayed cable, with each of its two arms holding to a neighboring hexon/penton capsomer and its vertex sitting atop a triplex. (B) A triangular facet of HCMV icosahedral reconstruction [14]. Three pUL32nt subunits form a “Δ”-shaped fortifying structure on every triplex, two of which (cyan and orange-red) form a “Λ”-shaped structure similar to that in MCMV, and the third (green) bridges the gap between edge and facet capsomers. The density of pM32nt/pUL32nt in Tf region was colored in blue as the densities in this region were smeared after imposing 3-fold symmetry during icosahedral reconstruction (See how this has been resolved in sub-particle reconstruction in S3 Fig).
Fig 7.
CATCs in MCMV and HCMV have conserved domain structures but divergent interacting interfaces.
(A) Green residues denote β-herpesvirus-conserved regions CR1 and CR2, while yellow residues denote the primate CMV-conserved cys region in pM32nt. (B) Structural alignment based on the Cα atoms of the two pM32nt conformers reveals a large degree of structural similarity between pM32nt-a and pM32nt-b in MCMV. (C) Residues involved in MCMV pM32nt-pM32nt (left panel) and HCMV pUL32nt-pUL32nt (right panel) interactions (whose atoms are within 3 Å from each other, as exemplified by pp150 dimers from triplex Te regions in MCMV and HCMV) are highlighted in blue, respectively. (D) The atomic models of triplex Td (gray) in HCMV and MCMV were aligned as a rigid body to show how their associated CATCs differ. Conformers b of CATC (i.e., pM32nt-b of MCMV and pUL32nt-b in HCMV) show major translational and rotational displacements (left panel) while conformers a (pM32nt-a and pUL32nt-a) only show minor rotational displacements (right panel) between HCMV and MCMV. Conformer c only exists in HCMV.
Fig 8.
pM32 does not bind to triplex Tc in MCMV.
(A) Zoom-in view of MCMV triplex Tc region. (B) The same region as shown in (A). Models of triplex and two pM32nt subunits from MCMV triplex Td region were docked as a rigid body into MCMV Tc region density by only fitting triplex structures into the map. Neighboring MCPs are colored individually while Tri1, Tri2A, Tri2B, pM32nt-a, pM32nt-b, and SCP are colored as in Fig 1D. (C) The right panel shows the atomic models which are presented using the same color scheme as in (B). The closest distances between pM32nt-a/pM32nt-b and their associated SCPs are shown in the left panels.
Fig 9.
Generation and analyses of M32-deletion MCMV mutant.
(A) Growth of the parental virus MCMVBAC (Smith), M32-deletion mutant ΔM32 and rescued mutant R-M32 in NIH 3T3 cells. NIH 3T3 cells were infected with each virus at a MOI of 0.5 PFU per cell. At 0, 1, 2, 3, 4, 5, and 6 days post-infection, we harvested the cells and culture media and determined the viral titers by plaque assays on NIH 3T3 cells. The error bars indicate the standard deviations based on triplicate experiments. (B-C) CryoEM images of the ΔM32 MCMV mutant. Fully enveloped particle and dense body are indicated by an open and a solid black arrow, respectively. (D) Radially colored surface representation of cryoEM reconstruction (~25 Å resolution) of the enveloped particles of ΔM32 MCMV, viewed along a 3-fold axis. The pentagon, triangle, and oval symbol denotes a 5-fold, 3-fold, and 2-fold axis, respectively. (E) Enlargement of a facet [triangle region in (D)] with pentons (Pen), hexons (C, E, and P) and triplexes (Ta, Tb, Tc, Td, Te, and Tf) indicated.