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HAPLESS2 (HAP2) is a broadly conserved, gamete-expressed transmembrane protein that was shown recently to be structurally homologous to viral class II fusion proteins, which initiate fusion with host cells via insertion of fusion loops into the host membrane. However, the functional conformation of the HAP2 fusion loops has remained unknown, as the reported X-ray structure of Chlamydomonas reinhardtii HAP2 lacked this critical region. Here, we report a structure-guided alignment that reveals diversification of the proposed HAP2 fusion loops. Representative crystal structures show that in flowering plants, HAP2 has a single prominent fusion loop projecting an amphipathic helix at its apex, while in trypanosomes, three small nonpolar loops of HAP2 are poised to interact with the target membrane. A detailed structure-function analysis of the Arabidopsis HAP2 amphipathic fusion helix defines key residues that are essential for membrane insertion and for gamete fusion. Our study suggests that HAP2 may have evolved multiple modes of membrane insertion to accommodate the diversity of membrane environments it has encountered during eukaryotic evolution., Author summary The fusion of gamete plasma membranes is the fundamental cellular event that brings two parental cells together to form a new individual, yet we know surprisingly little about this process at the molecular level. HAPLESS 2 (HAP2) is a conserved sperm plasma membrane protein that is essential for gamete fusion in a diverse array of eukaryotes. It was recently shown to share a common ancestor with viral proteins that drive fusion of the viral envelope with host membranes, but its mechanism of action remained elusive, since the reported structure did not resolve the proposed membrane interaction surface. Here, we report two new HAP2 structures revealing that HAP2 has evolved diverse membrane interaction surfaces. In the flowering plants, HAP2 uses an amphipathic helix that presents nonpolar residues to the target membrane; in trypanosomes, the membrane interaction surface comprises three shallow nonpolar loops.

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., Author Summary 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.