Structural basis of HSV-1 glycoproteins involved in viral entry. (IMAGE)
Caption
(A) Crystal structure of gD: The crystal structure of a monomer from the dimeric form of the gD ectodomain (PDB 2C36) is shown alongside AlphaFold3's prediction for the full-length gD dimer. The light purple surface highlights one monomer of the gD homodimer, allowing simultaneous visualization of the 3D surface and ribbon structure. The N-terminus (residues 6–38), responsible for nectin-1 or HVEM binding, is highlighted in cyan, while the pre-fusion domain (PFD, residues 260–285) involved in gD activation is highlighted in red.
(B, C) gD-receptor interactions: The crystal structures of gD bound to its receptors nectin-1 (B) and HVEM (C) illustrate receptor-binding mechanisms. AlphaFold3's predictions extend these structures, showing the interaction between the N-terminus of gD and the receptor, along with the positional changes of the PFD domain not observed in the resolved structures. In both cases, the N-terminus of gD (cyan) forms a loop containing all receptor-contact residues. Receptor binding displaces the PFD (red), allowing interactions with gH.
(D) Model of gD-Her2: A homodimeric model of gD engineered to target Her2. The anti-Her2 scFv (yellow) replaces the N-terminus of gD (residues 6–38).
(E) Structure of gH/gL: The HSV-2 gH/gL heterodimer (PDB 3M1C) is shown from a side view alongside AlphaFold3's prediction for the full-length HSV-1 gH/gL heterodimer.
(F) gH activation model: Upon binding to αVβ6 or αVβ8 integrins, gL dissociates, allowing gH to bind to receptor-activated gD. The ectodomain of gH interacts with the same surface previously occupied by gL. The PFD (red), along with an upstream 15-residue segment (green), forms a loop containing all contact residues (hydrogen bonds) for gH. These interactions between gH and gD were predicted using AlphaFold3.
(G) Model of gH-gB interactions: A model depicting the interaction between the cytoplasmic domain of gH and the gB trimer (PDB 5V2S). The pocket on the gBCTD surface is colored gray, with its base formed by residues T814 (yellow) and A851 (lime). According to the "clamp and wedge" model of gB activation, residue V831 (green) of gH wedges into this pocket, disrupting the inhibitory clamp and promoting the transition to post-fusion gB.
(H) Model of gH/gL-Her2: A heterodimeric model of gH/gL engineered to target Her2. The anti-Her2 scFv (yellow) is inserted at residue 23 of gH.
(I, J) Pre-fusion and post-fusion structures of gB: The crystal structures of the pre-fusion (I) and post-fusion (J) forms of the gB trimer are shown. The five domains of the gB ectodomain (DI to DV) are colored medium blue, orange, cyan, purple, and magenta, respectively. The fusion loops (red) in domain I form the trimer's legs, with the loops oriented toward the membrane. The box below shows the bottom of the pre-fusion gB trimer, and positively charged residues (177H, 178R, 258R, 263H, and 264R) and hydrophobic residues (174W, 179Y, 259V, and 262F) located in the fusion loops are highlighted. In the post-fusion gB structure, the membrane-proximal region (MPR, green), transmembrane region (TMR, blue), and cytoplasmic domain (CTD, dark gray) are visible.
(K) Insertable sites in gB: Distribution of sites in gB that tolerate insertions without disrupting surface expression or fusion activity. AlphaFold3's predictions highlight residues capable of accommodating large insertions, such as fluorescent proteins, including A42, A62, P81, A95, H100, R304, P361, R470, and P481 (shown as space-filled residues). Most insertion sites are located in the unstructured N-terminal region of gB (white).
(L) Model of gB-Her2: A post-fusion trimeric model of gB engineered to target Her2. The anti-Her2 scFv (yellow) is inserted at residue 42 of gB.
Credit
Yufang Zou, Juan Tao, Yingzheng Gao, Jixuan Wang, Pengfei Wang, Jingyuan Yan, Zuqing Nie, Dewei Jiang, Xinwei Huang
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