Structural basis for HCMV Pentamer recognition by neuropilin 2 and neutralizing antibodies

Abstract

Human cytomegalovirus (HCMV) encodes multiple surface glycoprotein complexes to infect a variety of cell types. The HCMV Pentamer, composed of gH, gL, UL128, UL130, and UL131A, enhances entry into epithelial, endothelial, and myeloid cells by interacting with the cell surface receptor neuropilin 2 (NRP2). Despite the critical nature of this interaction, the molecular determinants that govern NRP2 recognition remain unclear. Here, we describe the cryo-EM structure of NRP2 bound to Pentamer. The high-affinity interaction between these proteins is calcium dependent and differs from the canonical carboxyl-terminal arginine (CendR) binding that NRP2 typically uses. We also determine the structures of four neutralizing human antibodies bound to the HCMV Pentamer to define susceptible epitopes. Two of these antibodies compete with NRP2 binding, but the two most potent antibodies recognize a previously unidentified epitope that does not overlap the NRP2-binding site. Collectively, these findings provide a structural basis for HCMV tropism and antibody-mediated neutralization.

Fig. 1.. The interaction between NRP2 and Pentamer is calcium dependent.

(A) Primary sequence diagram of NRP2. The construct that was used for structural studies is colored orange. SS, signal sequence; MAM, meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu domain; TM, transmembrane domain; CD, cytoplasmic domain. (B) BLI sensorgram showing the absence of binding between Pentamer and NRP2 in the presence of 2 mM EDTA. (C) BLI sensorgram showing binding between Pentamer and NRP2 in the presence of 2 mM calcium. Data are shown as black lines, and best fit of a 1:1 binding model is shown as red lines. (D) BLI sensorgram showing binding between Pentamer and NRP2 a2b1b2 in the presence of 2 mM calcium. Data are shown as black lines, and best fit of a 1:1 binding model is shown as red lines.

Fig. 2.. The cryo-EM structure of the HCMV Pentamer bound by NRP2.

(A) Cryo-EM density is shown (left), with the Pentamer colored in shades of green, gray, and white and NRP2 colored orange. The atomic model of this complex (right) is shown as ribbons, with the NRP2 also represented by a transparent molecular surface. (B) Interface between the NRP2 a2 domain and UL128. UL128 is depicted as a transparent, green molecular surface with ribbons underneath, and NRP2 is shown as orange ribbons. Residues that are predicted to form critical contacts are shown as sticks. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and yellow, respectively. The single calcium atom is shown as a bright green sphere, with black dotted lines depicting the interaction with conserved coordinating residues. (C) Interface between the NRP2 b2 domain and the HCMV Pentamer. ULs 130 and 131A are shown as a transparent, green molecular surface with ribbons underneath, and NRP2 is shown as orange ribbons, with residues predicted to form critical contacts shown as sticks. Oxygen and nitrogen atoms are colored red and blue, respectively.

Fig. 3.. A subset of particles display a second copy of NRP2 bound to the C terminus of gL.

(A) Two-dimensional class averages of Pentamer bound by two copies of NRP2. (B) A ~4.2-Å cryo-EM reconstruction of Pentamer bound by two copies of NRP2 is shown as a transparent surface. Atomic models of each component are docked in, shown as ribbons. Both copies of NRP2 are colored orange, and Pentamer is colored white, except for gL, which is colored as a rainbow blue-to-red from the N terminus to the C terminus. The a1 domain from the gL-bound copy of NRP2 was excluded because it could not clearly be resolved in this reconstruction.

Fig. 4.. Composite of cryo-EM structures of Pentamer bound by four neutralizing human antibodies.

(A) Surface plasmon resonance sensorgrams showing binding of each of the four neutralizing Fabs, with data shown as black lines and the best fit of a 1:1 binding model shown as red lines. (B) The atomic models of two cryo-EM structures of antibodies bound to the HCMV Pentamer are superimposed on the basis of the position of the UL proteins. The Pentamer is shown as a molecular surface, colored according to Fig. 2, and Fabs are shown as ribbons surrounded by a transparent molecular surface. Fab 1-103 is colored blue, Fab 1-32 is colored gold, Fab 2-18 is colored purple, and Fab 2-25 is colored red. (C) CDRs from each Fab are shown as ribbons, and the Pentamer is shown as a transparent molecular surface with ribbons underneath. Predicted critical contact residues are shown as sticks. Fab 1-103 (top left) is colored blue, Fab 1-32 (bottom left) is colored gold, Fab 2-18 (top right) is colored purple, and Fab 2-25 (bottom right) is colored red. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and yellow, respectively.

Fig. 5.. Antibodies 2-18 and 2-25 potently neutralize HCMV without disrupting NRP2 binding.

(A) Neutralization curves are shown for each mAb based on inhibition of AD169rev-GFP infection in ARPE-19 cells. Inhibitory curves for both IgG and Fab are shown, with IgG shown in darker colors. (B) Post-attachment neutralization curves are shown for each mAb based on inhibition of AD169rev-GFP infection after adherence to ARPE-19 cells. (C) Relative IgG and Fab post-attachment neutralization potency. The reversed ratio of post-attachment IC₅₀ to standard IC₅₀ is plotted as a percentage for each IgG and Fab. (D) Neutralization potency of 2-18 IgG was evaluated against 12 clinical isolates and two laboratory-adapted HCMV strains in ARPE-19 cells. IC₅₀ values were calculated by nonlinear fit of the percentage of viral inhibition versus concentration (ng/ml). The curves used to calculate IC₅₀ values are shown in fig. S7. The neutralization results of mAbs 1-103, 1-32, and 2-25 against the same panel of HCMV strains have been reported previously (38). (E) Sensorgrams from a BLI-based competition experiment are displayed. NRP2 a1a2b1b2 was immobilized to a BLI sensor and dipped into Pentamer alone or Pentamer incubated with a molar excess of indicated Fab.

Fig. 6.. Pentamer-directed antibodies can neutralize HCMV via multiple mechanisms.

(A) Cryo-EM structures of NRP2-bound Pentamer and Fab-bound Pentamer are superimposed on the basis of the position of the UL proteins. Pentamer is shown as a molecular surface colored according to Fig. 2, Fabs are shown as ribbons, colored according to Fig. 4, and NRP2 is shown as orange ribbons surrounded by a transparent molecular surface. (B) Close-up views of each Fab-bound Pentamer are superimposed upon the NRP2-bound Pentamer. Both Fabs and NRP2 are shown as ribbons surrounded by a transparent molecular surface, while the Pentamer is shown as a solid molecular surface. NRP2 is colored orange, 1-103 is colored blue, 1-32 is colored gold, 2-18 is colored purple, and 2-25 is colored red.