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The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here we report the cryoEM structure of a coronavirus S glycoprotein in the postfusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the prefusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochemically characterized the molecular events associated with refolding of the metastable prefusion S glycoprotein to the postfusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The observed similarity between postfusion coronavirus S and paramyxovirus F structures demonstrates that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family., Competing Interests: The authors declare no conflict of interest.

Glycoprotein H (gH) is an envelope protein conserved in the Herpesviridae. Together with glycoprotein B (gB), the heterodimeric complex of gH and glycoprotein L (gL) mediates penetration and direct viral cell-to-cell spread. In herpes simplex and pseudorabies virus (PrV), coexpression of gH/gL, gB, and gD induces membrane fusion to form polykaryocytes. The recently determined crystal structure of a core fragment of PrV gH revealed marked structural similarity to other gH proteins (M. Backovic et al., Proc. Natl. Acad. Sci. U. S. A. 107:22635-22640, 2010). Within the membrane-proximal part (domain IV), a conserved negatively charged surface loop (flap) is flanked by intramolecular disulfide bonds. Together with an N-linked carbohydrate moiety, this flap covers an underlying patch of hydrophobic residues. To investigate the functional relevance of these structures, nonconservative amino acid substitutions were introduced by site-directed mutagenesis. The mutated proteins were tested for correct expression, fusion activity, and functional complementation of gH-deleted PrV. Several single amino acid changes within the flap and the hydrophobic patch were tolerated, and deletion of the glycosylation site had only minor effects. However, multiple alanine substitutions within the flap or the hydrophobic patch led to significant defects. gH function was also severely affected by disruption of the disulfide bond at the C terminus of the flap and after introduction of cysteine pairs designed to bridge the central part of the flap with the hydrophobic patch. Interestingly, all mutated gH proteins were able to complement gH-deleted PrV, but fusion-deficient gH mutants resulted in a pronounced delay in virus entry.

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. [ABSTRACT FROM AUTHOR]

Enveloped viruses enter cells by inducing fusion of viral and cellular membranes, a process catalyzed by a specialized membrane-fusion protein expressed on their surface. This review focuses on recent structural studies of viral fusion proteins with an emphasis on their metastable prefusion form and on interactions with neutralizing antibodies. The fusion glycoproteins have been difficult to study because they are present in a labile, metastable form at the surface of infectious virions. Such metastability is a functional requirement, allowing these proteins to refold into a lower energy conformation while transferring the difference in energy to catalyze the membrane fusion reaction. Structural studies have shown that stable immunogens presenting the same antigenic sites as the labile wild-type proteins efficiently elicit potently neutralizing antibodies, providing a framework with which to engineer the antigens for stability, as well as identifying key vulnerability sites that can be used in next-generation subunit vaccine design. A greater understanding of how viruses fuse with host cells, driven by recent advances in viral structural biology, is leading the way for new immunogen design and vaccine development. [ABSTRACT FROM AUTHOR]

This review covers the main features of the severe acquired respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, its interaction with the main entry receptor, the human angiotensin converting enzyme 2 (ACE2), and the subsequent membrane fusion step. The focus is on the structural organization of these proteins and mechanistic aspects of their interactions that lead to cytoplasmic release of the viral genome. The most potently neutralizing antibodies against SARS-CoV-2 were shown to interfere with the spike/ACE2 interaction. I thus also review the location and the potential impact of mutations in the spike protein observed in the variants of concern that emerged concomitantly with acquired immunity in the population after one year of virus circulation. Understanding how these interactions affect the spike/ACE2 interactions and the subsequent spike-protein-induced membrane fusion reaction is important to stay one step ahead of the virus evolution and develop efficient countermeasures.

Arenaviruses are important agents of zoonotic disease worldwide. The virions expose a tripartite envelope glycoprotein complex at their surface, formed by the glycoprotein subunits GP1, GP2 and the stable signal peptide. This complex is responsible for binding to target cells and for the subsequent fusion of viral and host-cell membranes for entry. During this process, the acidic environment of the endosome triggers a fusogenic conformational change in the transmembrane GP2 subunit of the complex. We report here the crystal structure of the recombinant GP2 ectodomain of the lymphocytic choriomeningitis virus, the arenavirus type species, at 1.8-Å resolution. The structure shows the characteristic trimeric coiled coil present in class I viral fusion proteins, with a central stutter that allows a close structural alignment with most of the available structures of class I and III viral fusion proteins. The structure further shows a number of intrachain salt bridges stabilizing the postfusion hairpin conformation, one of which involves an aspartic acid that appears released from a critical interaction with the stable signal peptide upon low pH activation. [ABSTRACT FROM AUTHOR]

Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class Ill viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor binding domain (RBD) of glycoprotein D fused to a truncated version of gH lacking its N- terminal domain. We report here the 2.1-Å resolution structure of the core fragment of gH present in this chimera, bound to the Fab fragment of a Pry gH-specific monoclonal antibody. The structure strongly complements the information derived from the recently reported structure of gH/gL from herpes simplex virus type 2 (HSV-2). Together with the structure of Epstein-Barr virus (EBV) gH/gL reported in parallel, it provides insight into potentially func- tional conserved structural features. One feature is the presence of a syntaxin motif, and the other is an extended "flap" masking a conserved hydrophobic patch in the C-terminal domain, which is closest to the viral membrane, The negative electrostatic surface potential of this domain suggests repulsive interactions with the lipid heads. The structure indicates the possible unmasking of an extended hydrophobic patch by movement of the flap during a re- ceptor-triggered conformational change of gH, exposing a hydro- phobic surface to interact with the viral membrane during the fusion process. [ABSTRACT FROM AUTHOR]

Foamy viruses (FVs) constitute a subfamily of retroviruses. Their envelope (Env) glycoprotein drives the merger of viral and cellular membranes during entry into cells. The only available structures of retroviral Envs are those from human and simian immunodeficiency viruses from the subfamily of orthoretroviruses, which are only distantly related to the FVs. We report the cryo-electron microscopy structures of the FV Env ectodomain in the pre-and post-fusion states, which unexpectedly demonstrate structural similarity with the fusion protein (F) of paramyxo- and pneumoviruses, implying an evolutionary link between the viral fusogens. We describe the structural features that are unique to the FV Env and propose a mechanistic model for its conformational change, highlighting how the interplay of its structural elements could drive membrane fusion and viral entry. The structural knowledge on the FV Env now provides a framework for functional investigations, which can benefit the design of FV Env variants with improved features for use as gene therapy vectors. [ABSTRACT FROM AUTHOR]

The article discusses the crystal structure of the Vesicular Stomatitis Virus Glycoprotein G. It is found that the virus has an atypical membrane fusion G showing a hydrogen ion concentration-dependent equilibrium between two forms at the virus surface. Membrane fusion is triggered during the transition from the high-pH to low-pH form. The structure of G in its low-pH form shows the classic hairpin conformation observed in all other fusion proteins in their postfusion conformation. G with gB of herpesviruses, describes a new family of fusion proteins exhibiting a new fusion module, an elongated β structure inserted in a pH domain and carrying two fusion loops. The structural organization of G is observed to be same as that of herpesvirus. The structural transition of G is significantly affected by mutations.

The retroviral envelope-derived proteins syncytin-1 and syncytin-2 (syn1 and syn2) drive placentation in humans by forming a syncytiotophoblast, a structure allowing for an exchange interface between maternal and fetal blood during pregnancy. Despite their essential role, little is known about the molecular mechanism underlying the syncytins' function. We report here the X-ray structures of the syn1 and syn2 transmembrane subunit ectodomains, featuring a 6-helix bundle (6HB) typical of the post-fusion state of gamma-retrovirus and filovirus fusion proteins. Contrary to the filoviruses, for which the fusion glycoprotein was crystallized both in the post-fusion and in the spring-loaded pre-fusion form, the highly unstable nature of the syncytins' prefusion form has precluded structural studies. We undertook a proline-scanning approach searching for regions in the syn1 6HB central helix that tolerate the introduction of helix-breaker residues and still fold correctly in the pre-fusion form. We found that there is indeed such a region, located two α-helical turns downstream a stutter in the central coiled-coil helix - precisely where the breaks of the spring-loaded helix of the filoviruses map. These mutants were fusion-inactive as they cannot form the 6HB, similar to the "SOSIP" mutant of HIV Env that allowed the high-resolution structural characterization of its labile pre-fusion form. These results now open a new window of opportunity to engineer more stable variants of the elusive pre-fusion trimer of the syncytins and other gamma-retroviruses envelope proteins for structural characterization. Image 1 • A typical retroviral γ-type Env protein 6-helix bundle in post-fusion syncytins. • An extensive ionic interactions network correlates with higher stability of syn1. • Spring-loaded pre-fusion form hinted by structural homology with filoviruses. • Helix-breaking residues in the central coiled-coil allow folding in pre-fusion form. [ABSTRACT FROM AUTHOR]

Conserved across the family Herpesviridae, glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomains of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the pseudorabies virus (PrV) gB ectodomain, revealing a typical class III postfusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct subregions of the lipid bilayer and their roles in membrane interactions. We tested 15 gB variants for the ability to bind to liposomes and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275, and Tyr276, which were essential for liposome binding and for fusion in cellular and viral contexts, form a continuous hydrophobic patch at the gB trimer surface. Together with results reported for other alphaherpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192, and Tyr276 form a rim that inserts into the more superficial interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gBs from beta- and gammaherpesviruses suggests that this membrane interaction model is valid for gBs from all herpesviruses. [ABSTRACT FROM AUTHOR]

Membrane fusion is indispensable for entry of enveloped viruses into host cells. The conserved core fusion machinery of the Herpesviridae consists of glycoprotein B (gB) and the gH/gL complex. Recently, crystal structures of gH/gL of herpes simplex virus 2 (HSV-2) and Epstein-Barr virus and of a core fragment of pseudorabies virus (PrV) gH identified four structurally conserved gH domains. To investigate functional conservation, chimeric genes encoding combinations of individual domains of PrV and herpes simplex virus 1 (HSV-1) gH were expressed in rabbit kidney cells, and their processing and transport to the cell surface, as well as activity in fusion assays including gB, gD, and gL of PrV or HSV-1, were analyzed. Chimeric gH containing domain I of HSV-1 and domains II to IV of PrV exhibited limited fusion activity in the presence of PrV gB and gD and HSV-1 gL, but not of PrV gL. More strikingly, chimeric gH consisting of PrV domains I to III and HSV-1 domain IV exhibited considerable fusion activity together with PrV gB, gD, and gL. Replacing PrV gB with the HSV-1 protein significantly enhanced this activity. A cell line stably expressing this chimeric gH supported replication of gH-deleted PrV. Our results confirm the specificity of domain I for gL binding, demonstrate functional conservation of domain IV in two alphaherpesviruses from different genera, and indicate species-specific interactions of this domain with gB. They also suggest that gH domains II and III might form a structural and functional unit which does not tolerate major substitutions. [ABSTRACT FROM AUTHOR]

Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-prolinecysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure shows that proline438 induces bending at the end of an alpha-helix, thereby placing cysteine404 and cysteine439 in juxtaposition to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution, we constructed different gH chimeras carrying proline or serine at position 438 in gH derived from either PrV strain Kaplan or strain Bartha. Mutants expressing gH with serine438 showed reduced fusion activity in transient-fusion assays and during infection, with delayed penetration kinetics and a small-plaque phenotype which indicates that proline438 is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine404 to serine. Although PrV expressing gHC404S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine438 to proline in gH of strain Bartha enhanced cell-to-cell spread and penetration kinetics, but restoration of full activity required additional alteration of aspartic acid to valine at position 59. [ABSTRACT FROM AUTHOR]

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble 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 cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses. • Atomic model of the hantavirus square surface glycoprotein lattice • Organization of the homotetrameric Gn base at atomic resolution • Control mechanism of Gc fusion loop exposure exerted by Gn on the spikes • Evolutionary links between alphaviruses and hantaviruses revealed by the glycoproteins X-ray structures of the hantavirus surface glycoprotein lattice reveal a built-in mechanism controlling envelope glycoprotein membrane insertion and provide important information for development of immunogen protection against these deadly viruses. [ABSTRACT FROM AUTHOR]

Summary The cellular fusion protein HAP2, which is structurally homologous to viral class II fusion proteins, drives gamete fusion across several eukaryotic kingdoms. Gamete fusion is a highly controlled process in eukaryotes, and is allowed only between same species gametes. In spite of a conserved architecture, HAP2 displays several species-specific functional regions that were not resolved in the available X-ray structure of the green alga Chlamydomonas reinhardtii HAP2 ectodomain. Here we present an X-ray structure resolving these regions, showing a target membrane interaction surface made by three amphipathic helices in a horseshoe-shaped arrangement. HAP2 from green algae also features additional species-specific motifs inserted in regions that in viral class II proteins are critical for the fusogenic conformational change. Such insertions include a cystine ladder-like module evocative of EGF-like motifs responsible for extracellular protein-protein interactions in animals, and a mucin-like region. These features suggest potential HAP2 interaction sites involved in gamete fusion control. Graphical Abstract Highlights • Unprecedented organization of amphipathic α helices in the algal HAP2 fusion loops • An inserted EGF-like motif suggests a potential algal-specific fusion control site • An adjacent mucin-like region potentially modulates algal-specific interactions • Inter-chain stem/domain II interactions stabilize the post-fusion hairpin conformation Baquero et al. report the structure of the HAP2 gamete fusion protein of the Chlamydomonas reinhardtii alga, describing key elements required to bridge two opposite-type gametes and drive their fusion. Mutagenesis of these elements confirmed their membrane-interaction function. Additional specific HAP2 protein-protein interaction sites are described as potential fusion controls. [ABSTRACT FROM AUTHOR]