Fig 1.
In situ structures of two distinct conformations of VSV G.
(A) A 10Å-thick slab from the tomogram showing bullet-shaped VSV virions. (B) The VSV particle indicated by red arrow in (A), with a yellow arrow pointing to long-form G and a yellow arrow head pointing to short-form G. (C~F) Subtomographic average of the long-form G densities, whose ectodomain matches the crystal structure of G in postfusion conformation. The subtomographic average of the long-form G densities is shown either as shaded surfaces viewed from side (C) and top (D), or as semi-transparent gray (E) fitted with crystal structure of the G trimer (ribbon) in the postfusion conformation (PDB: 5I2M) [20]. For clarity, one of the subunits is shown alone in (F) with five domains (DI~DV) indicated. (G~J) Subtomographic average of the short-form G densities, whose ectodomain matches the crystal structure of G in prefusion conformation. The subtomographic average of the short-form G densities is shown either as shaded surfaces viewed from side (G) and top (H), or as semi-transparent gray (I) fitted with crystal structure of the G trimer (ribbon) in the prefusion conformation (PDB: 5I2S) [21]. For clarity, one of the subunits is shown alone in (J) with five domains (DI~DV) indicated.
Fig 2.
(A) A 10Å-thick slab from the tomogram of S2 Movie showing a digital slice of various HCMV particles, each comprising an outer envelope layer, an intermediate tegument compartment, and an icosahedral capsid inside. Though all wrapped a pleomorphic glycoprotein-decorated envelope, these particles differ inside: each virion (yellow arrows) containing a C capsid (with DNA genome) and non-infectious enveloped particle (NIEP) either an A capsid (empty, blue arrow), or a B capsid (containing scaffolding protein but no DNA, red arrows). (B) A zoom-in slice of a C capsid, with dsDNA duplexes resolved among the fingerprint-like pattern of the genome. (C) A slice from the particle indicated by the cyan arrow in (A) showing the unique portal complex (arrow) at one of the 12 vertices. (D) A zoom-in envelope region of the particle indicated by a yellow arrow in (A) showing the two resolved leaflets of the lipid bilayer envelope (inset: the enlarged boxed region). The side of the boxes in B-D is 120 nm.
Fig 3.
In situ structures of gB in the “postfusion” and prefusion conformations.
(A) A representative virion showing various glycoprotein densities on its envelope. (B) Identifications of the columnar tree-shaped (red box) and Christmas tree-shaped (yellow box) glycoprotein densities that both match the expected volume of gB (see main text). Insets are the enlargements of the two forms with their corresponding shape schematic. (C~H) Sub-tomographic average of the columnar tree-shaped glycoprotein densities, whose ectodomain matches the crystal structure of gB ectodomain in the postfusion conformation (PDB: 5CXF) [16]. The subtomographic average of the columnar-shaped densities (yellow) and segmented membrane bilayer (blue, from I) are shown either as shaded surfaces viewed from side (C), top (D) and slanted bottom (E), or as semi-transparent gray fitted with the gB ectodomain trimer crystal structure (ribbon) at the postfusion conformation (PDB: 5CXF) [16] (F). Two subunits of the gB trimer crystal structure are shown as pink and gray surfaces, while the third subunit as ribbons with its domains colored as in [16] and its transmembrane helix as brown cylinder and the C-terminal flexible endodomain as a swinging dotted lines (G). For clarity, the third subunit is shown alone in (H) with five domains (DI~DV) indicated. (I~K) Sub-tomographic average of the Christmas tree-shaped densities (yellow) and associated membrane bilayer (blue) viewed from side (I), top (J) and slanted bottom (K).
Fig 4.
Schematic illustration of the full-length HCMV gB.
(A) Mapping of domains to the full-length HCMV gB primary sequence (SS = signal sequence, MPR = membrane proximal region, TM = transmembrane domain, Endo = endodomain). Upper inset: in the prefusion (Pre) conformation, the sequence of the central helix in DIII resolved in the postfusion gB crystal structure (Post) is predicted to fold into two helices joined by a short loop around residues 498–500. Lower inset: predicted secondary structures of the sequence encompassing the MPR, TM and endodomain of gB in the prefusion conformation. (B) Helical wheel diagram of the first 15 amino acids of MPR (sequence in red dashed box in (A)), showing one side with a cluster of hydrophobic amino acids.
Fig 5.
Domain fitting for prefusion gB.
(A~C) Domain fitting for DI. (A) Scores of 28 models for DI. Models are ranked in ascending order of scores. Red dot indicates the model of highest score. Dots with green and orange circles indicate the models of medium and low score, respectively. The DI models, superimposed in the subtomographic average of the Christmas tree-shaped density (semi-transparent gray) and viewed from side and top, are shown in green and orange dashed boxes next to the corresponding dots, respectively. (B, C) DI structure, indicated as red dot in (A), is superimposed in the subtomographic average of the Christmas tree-shaped density (semitransparent gray), viewed from side (B) and top (C). (D) VSV G domain rearrangement of crystal monomer structure from prefusion (left, PDB: 2J6J) [21] to postfusion (right, PDB: 2CMZ) [21]. The red dotted lines represent the unresolved domain DV between transmembrane helix and the ectodomain. (E) the crystal structure of one protomer of the postfusion HCMV gB ectodomain (left, PDB: 5CXF) [16] is shown as ribbon next to the predicted prefusion gB structure (right) with domains arranged according to those in the prefusion VSV G. α4 and α5 represent the long central helix and the following short helix in DIII in postfusion gB structure. α4a and α4b represent the two helix breaking from α4. Helices are labeled as in [16]. (F, G) The predicted prefusion gB structure shown in (E, right) is superposed with two other symmetric copies (gray ribbon) in the subtomographic average of the Christmas tree-shaped density (semi-transparent gray), viewed from side (F) and top (G). (H, I) The MDFF-simulated prefusion gB structure is superimposed with two other symmetric copies (gray ribbon) in the subtomographic average of the Christmas tree-shaped density (semi-transparent gray), viewed from side (H) and top (I). The epitopes of HSV-1 antibodies SS55/SS56 and R240 are DI and fusion loop 2 of HSV-1 gB, respectively ([26]); the corresponding locations of these two epitopes in our domain model of HCMV gB are indicated.
Fig 6.
In situ structure of gH/gL complex adjacent to prefusion gB.
(A) A series of slices in a tomogram showing a HCMV particle at different sections. (B) A 3D surface view from the HCMV particle in (A) with subtomographic average of prefusion and “postfusion” gB and gH/gL placed back on the viral envelope segmented from the tomogram. Black arrow indicates the gB/gH/gL complex. Unidentified glycoprotein densities are indicated as rods. Tegument proteins are shown as cyan densities. The recently published icosahedral reconstruction of capsid [11] was low-passed to 10Å, radially colored and placed back in its location. (C~F) The subtomographic average (C) showing a putative gH/gL complex adjacent to prefusion gB. The subtomographic average is also shown fitted with crystal structure of gH/gL (ribbon) (PDB: 5VOB) [17] in semitransparent surface viewed from side (D), top (E) and bottom (F).
Fig 7.
Schematic illustration of conformation changes of gB during membrane fusion.
(A, B) Subtomographic averages of prefusion gB (A) with domains illustrated as in Fig 3 and “postfusion” gB (B) with domains colored as in [16]. (C) A working model of gB conformational change during membrane fusion. In step 1, destabilization of the endodomain of prefusion gB either by cytotail conformational changes following gH/gL receptor-binding or by other means (e.g., mechanical stress such as high-speed centrifugation during viral purification) triggers DI and DII to reorient, exposing the fusion loops on DI. Subsequently, the exposed fusion loops could make contact either with cell membrane in close proximity (in the case of receptor binding) (step 2) or with viral membrane. Finally (step 3), DV refolds into an extended form, transforming gB into its “postfusion” conformation: in the presence of cell membrane, the C-terminal part and the fusion loops come together and the membranes fuse; in the absence of cell membrane, the exposed fusion loops insert into the viral membrane.