The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control Mechanism

Abstract

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. The hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further associate into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head, and of the homotetrameric Gn base. Docking them into an 11.4 Å resolution cryo-electron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and provided unprecedented detail on the surface organization of these pleomorphic virions. Our results, which further revealed an in-built mechanism controlling Gc membrane-insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses Man scrip

Molecular rationale for antibody-mediated targeting of the hantavirus fusion glycoprotein

“…PUUV Gc Gc−P-4G2 fits well within the cryo-ET reconstruction, where the crystallographically observed conformation of domain III accurately matches a feature of the glycoprotein spike ( Figure 5C ), further supporting that PUUV Gc has crystallized in the pre-fusion conformation presented on the mature virion. Additionally, this fitting confirms the location of Gc in the density spanning from the membrane-distal globular lobes to the viral membrane, as we and others have proposed previously ( Li et al, 2016 ; Serris et al, 2020 ). The fitting locates domain III of the Gc adjacent to the membrane, allowing the transmembrane region of the C-terminus to be linked to the virion envelope ( Figures 5A and 2A ), and places the fusion loop from Gc domain II into membrane-distal lobe density in close contact with Gn ( Figure 5A and B ), in a manner similar to that observed for other class II fusion proteins and their cognate accessory proteins ( Halldorsson et al, 2018 ; Voss et al, 2010 ).…”

“…Cryo-ET of purified PUUV VLPs, in tandem with sub-tomogram averaging, revealed that the VLP surface displays the expected Gn−Gc spike architecture that can form locally ordered lattices, as observed in native hantavirions ( Figure 4—figure supplement 1 ). Consistent with previous studies of hantavirus ultrastructure and Gc oligomerization ( Bignon et al, 2019 ; Li et al, 2016 ), breakpoints exist in the (Gn−Gc) 4 lattices ( Huiskonen et al, 2010 ; Li et al, 2016 ; Serris et al, 2020 ). Previous studies have suggested that contacts between (Gn−Gc) 4 spikes are mediated by Gc homo-dimers ( Bignon et al, 2019 ; Huiskonen et al, 2010 ; Li et al, 2016 ), while lattice breaks are expected to display Gc (‘lattice-free Gc’) molecules that are not integrated within the lattice assembly through Gc homo-dimer interactions.…”

“…Consistent with the hypothesis that mAb P-4G2 targets a pre-fusion conformation of PUUV Gc representative of that displayed on the native virion surface, structure overlay analysis reveals that PUUV Gc from the Gc–P-4G2 complex (PUUV Gc Gc–P-4G2 ) is highly similar to the recently reported pre-fusion state of Andes virus (ANDV) Gc ( Serris et al, 2020 ), and is distinct from the known PUUV Gc post-fusion structure ( Willensky et al, 2016 ; Figure 3 and Figure 3—figure supplement 1 ). Notably, the conformations of domain III from both the recently determined ANDV Gc ( Serris et al, 2020 ) and our PUUV Gc contrast those observed across hantavirus Gc structures ( Figure 3—figure supplement 1 ), suggesting that this region is likely flexible in the absence of the stabilizing environment of the glycoprotein lattice or crystal environments. Additionally, the conformation of PUUV Gc Gc–P-4G2 closely resembles class II fusion protein pre-fusion states, such as those observed for RVFV Gc (PDB 4HJ1) and ZIKV E (PDB 5IRE) ( Figure 3—figure supplement 1 ).…”

“… ( A ) The conformation of PUUV Gc Gc - P-4G2 is distinct from ( B ) the previously reported PUUV Gc post-fusion structure (PDB 5J9H) ( Willensky et al, 2016 ), and from ( C ) the intermediate configuration of HTNV Gc (PDB 5LJY) ( Guardado-Calvo et al, 2016 ), and aligns more closely with ( D ) the recently published prefusion Andes virus (ANDV) Gc, crystallized in complex with cognate ANDV Gn (PDB 6Y5F) ( Serris et al, 2020 ), and ( E ) the experimentally confirmed pre-fusion conformations of RVFV Gc (PDB 4HJ1) ( Dessau and Modis, 2013 ) and ( F ) Zika E (PDB 5IRE) ( Sirohi et al, 2016 ). Cartoon representations of the structure are colored according to domain boundaries (red, yellow, and blue for domains I, II, and III, respectively).…”

“…The Gc adopts a three-domain (domains I−III) class II fusion protein architecture and is postulated to associate closely with the Gn, linking adjacent Gn tetramers ( Guardado-Calvo et al, 2016 ; Hepojoki et al, 2010 ; Li et al, 2016 ; Willensky et al, 2016 ). The Gn has been shown to shield the fusion loop resident in domain II of the Gc, in a manner similar to that proposed in phleboviruses and alphaviruses ( Allen et al, 2018 ; Guardado-Calvo et al, 2016 ; Guardado-Calvo and Rey, 2017 ; Halldorsson et al, 2018 ; Li et al, 2016 ; Serris et al, 2020 ; Voss et al, 2010 ). Following host cell binding and endocytotic uptake of the virus, acidification in endosomal compartments results in at least partial dissolution of the Gn−Gc lattice ( Acuña et al, 2015 ; Hepojoki et al, 2010 ; Rissanen et al, 2017 ).…”