Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single “fusion loop”. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal “tail” that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens. Hantaviruses belong to the Bunyaviridae family of enveloped viruses. This family englobes in total five established genera: Tospovirus (infecting plants), and Phlebovirus, Orthobunyavirus, Nairovirus and Hantavirus infecting animals, some of which cause serious disease in humans. An important characteristic of the hantaviruses is that they are not transmitted to humans by arthropod vectors, as those from the other genera, but by direct exposure to excretions from infected small mammals. As all enveloped viruses, they require the activity of a membrane fusogenic protein, Gc, for entry into their target cells. Our structural analysis of the hantavirus fusion protein Gc led to the identification of a conserved pattern of cysteines involved in disulfide bonds stabilizing the Gc fold. This motif is matched exclusively by all of the available bunyavirus Gc sequences in the database, with the notable exception of phlebovirus Gc, which appears closer in structure to the fusion proteins of other families of arthropod-borne viruses, such as the flaviviruses and alphaviruses. This analysis further suggests mechanistic similarities with hantaviruses in the fusion mechanism of viruses in the remaining three most closely related bunyavirus genera, which we propose belong to a new separate sub-class of fusion proteins with a multipartite membrane targeting region.

The hantavirus envelope glycoproteins Gn and Gc mediate virion assembly and cell entry, with Gc driving fusion of viral and endosomal membranes. Although the X-ray structures and overall arrangement of Gn and Gc on the hantavirus spikes are known, their detailed interactions are not. Here we show that the lateral contacts between spikes are mediated by the same 2-fold contacts observed in Gc crystals at neutral pH, allowing the engineering of disulfide bonds to cross-link spikes. Disrupting the observed dimer interface affects particle assembly and overall spike stability. We further show that the spikes display a temperature-dependent dynamic behavior at neutral pH, alternating between ‘open’ and ‘closed’ forms. We show that the open form exposes the Gc fusion loops but is off-pathway for productive Gc-induced membrane fusion and cell entry. These data also provide crucial new insights for the design of optimized Gn/Gc immunogens to elicit protective immune responses.

Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, G_N and G_C, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as G_N and G_C assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (G_C) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows G_C as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, G_C was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV G_C structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens. Hantaviruses (family: Bunyaviridae) encompass pathogens responsible to serious human diseases and economic burden worldwide. Following endocytosis, these enveloped RNA viruses are directed to an endosomal compartment where a sequence of pH-dependent conformational changes of the viral envelope glycoproteins mediates the fusion between the viral and endosomal membranes. The lack of high-resolution structural information for the entry of hantaviruses impair our ability to rationalize new treatments and prevention strategies. We determined the three-dimensional structure of a glycoprotein C from Puumala virus (PUUV) using X-ray crystallography. The two structures (at pH 6.0 and 8.0) were determined to 1.8 Å and 2.3 Å resolutions, respectively. Both structures reveal a class II membrane fusion protein in its post-fusion trimeric conformation with novel structural features in the trimer assembly and stabilization. Our structures suggest that neutralizing antibodies against G_C target its conformational changes as inhibition mechanism and highlight new molecular targets for hantavirus-specific membrane fusion inhibitors. Furthermore, combined with the available structures of other class II proteins, we remodeled the evolutionary relationships between virus families encompassing these proteins.

Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.

The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. Yet, little is known about the specific mechanism that triggers Gc fusion activation and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles (VLPs) with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.