Structural basis for HCMV Pentamer receptor recognition and antibody neutralization

Abstract

Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.

Fig. 1.. Structure of HCMV Pentamer gHgLUL128-131A bound to NRP2.

(A) HCMV Pentamer from two different strains (Merlin and VR1814) selectively a interacts with NRP2 and not NRP1, as previously described (5). (B) Schematic domain organization of human NRP2. (C) Front view of HCMV Pentamer complex (shown in ribbon) bound to NRP2 a2b1b2 domains (shown in surface representation). Fabs are not rendered for clarity. (D) Close-in view of HCMV Pentamer distal region showing NRP2 a1a2b1b2 domains, UL128-131A, gL, and the gH N terminus. NRP2 a1 appears to interact loosely with HCMV Pentamer, is not well resolved in the cryo-EM map, and is not modeled in the structure. Fabs are not rendered for clarity. (E) Close-in view of HCMV Pentamer distal region as described in (D) with highlighted sites of interaction between Pentamer and NRP2 a2 domain (site 1). Residues in bold represent reverse-charge mutations. (F) Close-in view of HCMV Pentamer distal region as described in (D) with highlighted sites of interaction between Pentamer and NRP2 b2 domain (site 2). Residues in bold represent reverse-charge mutations. (G) Close-in view of HCMV Pentamer distal region as described in (D) with highlighted surface interaction area to NRP2. (H) Summarized binding affinities of Pentamer to NRP2 WT or single-site point mutations (site 1: N172R, M253E, and A254E; site 2: Y458R and L459R). Kinetic parameters are representative of two independent assays.

Fig. 2.. Structure of HCMV Pentamer gHgLUL128-131A bound to THBD.

(A) Schematic domain organization of human THBD. (B) Top and front view of dimeric HCMV Pentamer complex (shown in ribbon) bound to THBD lectin domain (shown in surface representation). Close-in view of HCMV Pentamer 1 distal region showing THBD lectin domains, UL128-131A, and gL with highlighted sites of interaction between Pentamer 1 and THBD lectin domain. Close-in view of HCMV Pentamer 2 distal region showing THBD lectin domains, UL128-131A, and gL with highlighted sites of interaction between Pentamer 2 and THBD lectin domain. (C) Close-in view of HCMV Pentamer 1 distal region as described in (C) with highlighted surface interaction area to THBD. (D) Close-in view of HCMV Pentamer 2 distal region as described in (D) with highlighted surface interaction area to THBD.

Fig. 3.. THBD is a functional HCMV receptor and competes with NRP2 for HCMV Pentamer binding.

(A) HUVEC or ARPE-19 cells were infected for 48 hours with VR1814 virus that had been preincubated with different concentrations of an anti-NRP2 antibody (red open circles), soluble recombinant CD46 (blue full circle), NRP2 (red full circle), THBD (orange full circle), a mix of recombinant NRP2 with THBD proteins (violet full circle), or a mix of anti-NRP2 with recombinant THBD proteins (violet full circle). The percentage of infected cells plotted against antibody or protein concentration is shown. The data shown are the means of three independent experiments ± SD. (B) Histogram representing the percentage of infection of HAP-1 cells WT or NRP2-KO that have been transduced with either empty lentivirus vector, lentivirus vector encoding CD46, or lentivirus vector encoding THBD. The percentage of maximum infection (geometric means of four independent experiments ± SD) is shown. n.s., not significant. (C) Overlay of HCMV Pentamer in complex with NRP2 and THBD. The a1 domain is shown in surface representation. (D) Binding of HCMV Pentamer to NRP2-Fc WT and increasing concentrations of THBD starting at equimolar ratio or addition of 100-fold excess of transforming growth factor β receptor 3 (TGFβR3) as a control. (E) Overlay between the HCMV Pentamer–NRP2 dimer cryo-EM map and the dimeric structure of HCMV Pentamer–THBD. (F) HCMV Pentamer dimerization interface mediated by UL128. (G) Close-in view of the dimeric interaction interface mediated by UL128 [top view relative to view in (G)].

Fig. 4.. Structural basis for HCMV Pentamer neutralization.

(A) Front view of a composite structure of HCMV Pentamer complex (shown in ribbon) bound to the Pentamer-specific neutralizing antibodies 2C12, 7I13, and 8I21 (shown in surface representation) and the gH-specific neutralizing antibodies 13H11 and MSL-109 (shown in surface representation). (B and C) Close-in view of HCMV Pentamer distal region showing 2C12, UL128-131A, and gL with highlighted sites of interaction between Pentamer and 2C12. (D) Highlighted interaction interface between Pentamer and 2C12 or 7I13. (E) Close-in view of HCMV Pentamer distal region showing 2C12, UL128-131A, and gL 2C12 and 7I13. (F to I) Close-in view of HCMV Pentamer distal region showing 7I13, UL128-131A, and gL with highlighted sites of interaction between Pentamer and 7I13. (J) Overlay between HCMV Pentamer–NRP2 and HCMV Pentamer–2C12. (K) Overlay between HCMV Pentamer–THBD and HCMV Pentamer–7I13. (L) Binding of HCMV Pentamer to NRP2-Fc WT upon addition of the neutralizing Fabs 2C12, 7I13, 8I21, MSL-109, or 13H11. (M) Binding of HCMV Pentamer to THBD-Fc WT upon addition of the neutralizing Fabs 2C12, 7I13, 8I21, MSL-109, or 13H11.

Fig. 5.. Model for HCMV Pentamer neutralization and Pentamer-mediated cell entry.

Receptor binding and possibly Pentamer dimerization could mediate receptor clustering and facilitate a high affinity and stable tethering of the viral membrane with the host cell membrane. Membrane tethering would trigger the transition of gB from its pre- to post-fusion conformation and initiate membrane fusion. A similar mechanism involving the Trimer, in the absence of receptor multimerization, has also been described (16).