A Molecular-Level Account of the Antigenic Hantaviral Surface

Abstract

Hantaviruses, a geographically diverse group of zoonotic pathogens, initiate cell infection through the concerted action of Gn and Gc viral surface glycoproteins. Here, we describe the high-resolution crystal structure of the antigenic ectodomain of Gn from Puumala hantavirus (PUUV), a causative agent of hemorrhagic fever with renal syndrome. Fitting of PUUV Gn into an electron cryomicroscopy reconstruction of intact Gn-Gc spike complexes from the closely related but non-pathogenic Tula hantavirus localized Gn tetramers to the membrane-distal surface of the virion. The accuracy of the fitting was corroborated by epitope mapping and genetic analysis of available PUUV sequences. Interestingly, Gn exhibits greater non-synonymous sequence diversity than the less accessible Gc, supporting a role of the host humoral immune response in exerting selective pressure on the virus surface. The fold of PUUV Gn is likely to be widely conserved across hantaviruses.

Graphical abstract

Figure 1

Crystal Structure of the Puumala Gn Ectodomain (A) A ribbon representation of Puumala (PUUV) Gn colored from blue (N terminus) to red (C terminus). N-linked glycans are shown as green sticks. (B) Domain schematic of PUUV glycoprotein precursor with the signal peptide (SP), ectodomain, transmembrane domain (TM), intravirion domain (IV), zinc finger (ZF), and WAASA signal peptidase cleavage site shown (produced with DOG; Ren et al., 2009). Y-shaped symbols designate N-linked glycosylation sites. The location of the additional putative N-linked glycosylation site at Asn235 in Hantaan virus (Lys243 in PUUV) is indicated in gray. See also Figures S1 and S2.

Figure 2

Organization of Tula Virion (A) Low-pass filtered computational section from a tomographic reconstruction of three Tula virus (TULV) virions. Gn-Gc glycoprotein spikes are indicated with arrowheads. Scale bar, 50 nm. (B) Inset from (A) showing a magnified view of the membranous region of one virion. Scale bar, 15 nm. (C) A TULV virion showing higher order architecture of reconstructed TULV Gn-Gc glycoprotein spikes (gray), prepared by mapping spike complexes onto the virion lipid bilayer envelope (cyan). Zoom-in panel (bottom) reveals the higher-order glycoprotein lattice of Gn-Gc spikes. (D and E) Top (D) and side (E) views of the 16-Å-resolution TULV Gn-Gc glycoprotein spike with the fitted crystal structure of PUUV Gn. (F) Schematic of (E) with dimensions and putative density assignments annotated. See also Figures S3–S5.

Figure 3

Mapping Functional Residues onto PUUV Gn Surface (A) PUUV Gn fitted into the TULV reconstruction, as in Figure 2, with zoom panel shown (bottom). (B) Mapping the antigenic surface of PUUV Gn. Predicted mAb 5A2 neutralizing epitopes are colored magenta and purple (A/A′ and B/B′, respectively). Patient sera-reactive epitopes are colored salmon. The antibody neutralization evasion site (D272V) is colored red. (C) Mapping sequence conservation onto PUUV Gn. Well-conserved (green), average (white), and variable (yellow) regions are shown. The conservation analysis was performed with Consurf (Ashkenazy et al., 2010) using the hantaviral sequences listed in the Figure S2 legend. See also Figures S2–S5.