Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro

ABSTRACT Enveloped viruses require viral fusion proteins to promote fusion of the viral envelope with a target cell membrane. To drive fusion, these proteins undergo large conformational changes that must occur at the right place and at the right time. Understanding the elements which control the stability of the prefusion state and the initiation of conformational changes is key to understanding the function of these important proteins. The construction of mutations in the fusion protein transmembrane domains (TMDs) or the replacement of these domains with lipid anchors has implicated the TMD in the fusion process. However, the structural and molecular details of the role of the TMD in these fusion events remain unclear. Previously, we demonstrated that isolated paramyxovirus fusion protein TMDs associate in a monomer-trimer equilibrium, using sedimentation equilibrium analytical ultracentrifugation. Using a similar approach, the work presented here indicates that trimeric interactions also occur between the fusion protein TMDs of Ebola virus, influenza virus, severe acute respiratory syndrome coronavirus (SARS CoV), and rabies virus. Our results suggest that TM-TM interactions are important in the fusion protein function of diverse viral families. IMPORTANCE Many important human pathogens are enveloped viruses that utilize membrane-bound glycoproteins to mediate viral entry. Factors that contribute to the stability of these glycoproteins have been identified in the ectodomain of several viral fusion proteins, including residues within the soluble ectodomain. Although it is often thought to simply act as an anchor, the transmembrane domain of viral fusion proteins has been implicated in protein stability and function as well. Here, using a biophysical approach, we demonstrated that the fusion protein transmembrane domains of several deadly pathogens—Ebola virus, influenza virus, SARS CoV, and rabies virus—self-associate. This observation across various viral families suggests that transmembrane domain interactions may be broadly relevant and serve as a new target for therapeutic development.

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Virus Membrane Fusion Proteins: Biological Machines that Undergo a Metamorphosis

Bioscience Reports ◽

10.1023/a:1010467106305 ◽

2000 ◽

Vol 20 (6) ◽

pp. 597-612 ◽

Cited By ~ 90

Author(s):

Rebecca Ellis Dutch ◽

Theodore S. Jardetzky ◽

Robert A. Lamb

Keyword(s):

Fusion Proteins ◽

Ebola Virus ◽

Transmembrane Domain ◽

Coiled Coil ◽

Fusion Peptide ◽

Proteolytic Cleavage ◽

Heptad Repeat ◽

Viral Fusion ◽

Hydrophobic Region ◽

Viral Fusion Proteins

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4–3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structuralelements in the processing and function of these viral fusion proteins is discussed.

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New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes

Viruses ◽

10.3390/v12040413 ◽

2020 ◽

Vol 12 (4) ◽

pp. 413 ◽

Cited By ~ 3

Author(s):

Mark A. Benhaim ◽

Kelly K. Lee

Keyword(s):

Membrane Fusion ◽

Conformational Changes ◽

Viral Entry ◽

Fusion Proteins ◽

Structural Changes ◽

Fusion Reaction ◽

Type I ◽

Viral Fusion ◽

Technological Advances ◽

Viral Fusion Proteins

Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Recent advances in structural and biophysical methodologies have enabled researchers to directly observe viral fusion proteins as they carry out their functions during membrane fusion. Here we review the structure and function of type I viral fusion proteins and mechanisms of protein-mediated membrane fusion. We highlight how recent technological advances and new biophysical approaches are providing unprecedented new insight into the membrane fusion reaction.

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Viral Membrane Fusion and the Transmembrane Domain

Viruses ◽

10.3390/v12070693 ◽

2020 ◽

Vol 12 (7) ◽

pp. 693 ◽

Cited By ~ 1

Author(s):

Chelsea T. Barrett ◽

Rebecca Ellis Dutch

Keyword(s):

Membrane Fusion ◽

Host Cell ◽

Fusion Proteins ◽

Transmembrane Domain ◽

Critical Role ◽

Viral Fusion ◽

Transmembrane Region ◽

Host Cell Membrane ◽

The Past ◽

Viral Fusion Proteins

Initiation of host cell infection by an enveloped virus requires a viral-to-host cell membrane fusion event. This event is mediated by at least one viral transmembrane glycoprotein, termed the fusion protein, which is a key therapeutic target. Viral fusion proteins have been studied for decades, and numerous critical insights into their function have been elucidated. However, the transmembrane region remains one of the most poorly understood facets of these proteins. In the past ten years, the field has made significant advances in understanding the role of the membrane-spanning region of viral fusion proteins. We summarize developments made in the past decade that have contributed to the understanding of the transmembrane region of viral fusion proteins, highlighting not only their critical role in the membrane fusion process, but further demonstrating their involvement in several aspects of the viral lifecycle.

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Reevaluating Herpes Simplex Virus Hemifusion

Journal of Virology ◽

10.1128/jvi.01615-10 ◽

2010 ◽

Vol 84 (22) ◽

pp. 11814-11821 ◽

Cited By ~ 20

Author(s):

Julia O. Jackson ◽

Richard Longnecker

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Fusion Protein ◽

Membrane Fusion ◽

Fusion Proteins ◽

Fusion Pore ◽

Viral Fusion ◽

Viral Fusion Protein ◽

Viral Fusion Proteins ◽

Simplex Virus

ABSTRACT Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.

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Purification and Crystallization Reveal Two Types of Interactions of the Fusion Protein Homotrimer of Semliki Forest Virus

Journal of Virology ◽

10.1128/jvi.78.7.3514-3523.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3514-3523 ◽

Cited By ~ 20

Author(s):

Don L. Gibbons ◽

Brigid Reilly ◽

Anna Ahn ◽

Marie-Christine Vaney ◽

Armelle Vigouroux ◽

...

Keyword(s):

Fusion Protein ◽

Fusion Proteins ◽

Semliki Forest Virus ◽

Three Dimensional ◽

Class Ii ◽

Class I ◽

Structural Basis ◽

Viral Fusion ◽

Purification Method ◽

Viral Fusion Proteins

ABSTRACT The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein.

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Low-pH induced conformational changes in viral fusion proteins: implications for the fusion mechanism

Journal of General Virology ◽

10.1099/0022-1317-76-7-1541 ◽

1995 ◽

Vol 76 (7) ◽

pp. 1541-1556 ◽

Cited By ~ 90

Author(s):

Y. Gaudin ◽

R. W. H. Ruigrok ◽

J. Brunner

Keyword(s):

Conformational Changes ◽

Fusion Proteins ◽

Low Ph ◽

Viral Fusion ◽

Viral Fusion Proteins

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Site-Directed Antibodies against the Stem Region Reveal Low pH-Induced Conformational Changes of the Semliki Forest Virus Fusion Protein

Journal of Virology ◽

10.1128/jvi.01054-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9599-9607 ◽

Cited By ~ 9

Author(s):

Maofu Liao ◽

Margaret Kielian

Keyword(s):

Fusion Protein ◽

Conformational Changes ◽

Fusion Proteins ◽

Semliki Forest Virus ◽

Transmembrane Domain ◽

Polyclonal Antibodies ◽

Class Ii ◽

Low Ph ◽

Protein Domain ◽

Domain Iii

ABSTRACT The E1 envelope protein of the alphavirus Semliki Forest virus (SFV) is a class II fusion protein that mediates low pH-triggered membrane fusion during virus infection. Like other class I and class II fusion proteins, during fusion E1 inserts into the target membrane and rearranges to form a trimeric hairpin structure. The postfusion structures of the alphavirus and flavivirus fusion proteins suggest that the “stem” region connecting the fusion protein domain III to the transmembrane domain interacts along the trimer core during the low pH-induced conformational change. However, the location of the E1 stem in the SFV particle and its rearrangement and functional importance during fusion are not known. We developed site-directed polyclonal antibodies to the N- or C-terminal regions of the SFV E1 stem and used them to study the stem during fusion. The E1 stem was hidden on neutral pH virus but became accessible after low pH-triggered dissociation of the E2/E1 heterodimer. The stem packed onto the trimer core in the postfusion conformation and became inaccessible to antibody binding. Generation of the E1 homotrimer on fusion-incompetent membranes identified an intermediate conformation in which domain III had folded back but stem packing was incomplete. Our data suggest that E1 hairpin formation occurs by the sequential packing of domain III and the stem onto the trimer core and indicate a tight correlation between stem packing and membrane merger.

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Conserved Leucines in N-Terminal Heptad Repeat HR1 of Envelope Fusion Protein F of Group II Nucleopolyhedroviruses Are Important for Correct Processing and Essential for Fusogenicity

Journal of Virology ◽

10.1128/jvi.01885-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2437-2447 ◽

Cited By ~ 4

Author(s):

Gang Long ◽

Xiaoyu Pan ◽

Just M. Vlak

Keyword(s):

Fusion Protein ◽

Fusion Proteins ◽

Leucine Zipper ◽

Fusion Peptide ◽

Class I ◽

Heptad Repeat ◽

Viral Fusion ◽

F Protein ◽

Viral Fusion Proteins ◽

Budded Virus

ABSTRACT The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F1. Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.

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Identification of an N-Terminal Trimeric Coiled-Coil Core within Arenavirus Glycoprotein 2 Permits Assignment to Class I Viral Fusion Proteins

Journal of Virology ◽

10.1128/jvi.00008-06 ◽

2006 ◽

Vol 80 (12) ◽

pp. 5897-5907 ◽

Cited By ~ 96

Author(s):

Bruno Eschli ◽

Katharina Quirin ◽

Alexander Wepf ◽

Jacqueline Weber ◽

Rolf Zinkernagel ◽

...

Keyword(s):

Fusion Protein ◽

Fusion Proteins ◽

Gel Filtration ◽

Class I ◽

Spherical Particles ◽

Viral Fusion ◽

Viral Fusion Protein ◽

Helical Peptide ◽

Viral Fusion Proteins ◽

Α Helix

ABSTRACT The lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) consists of the transmembrane subunit GP-2 and the receptor binding subunit GP-1. Both are synthesized as one precursor protein and stay noncovalently attached after cleavage. In this study, we determined the oligomeric state of the LCMV GP and expressed it in two different conformations suitable for structural analysis. Sequence analysis of GP-2 identified a trimeric heptad repeat pattern containing an N-terminal α-helix. An α-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second α-helical peptide corresponding to a predicted C-terminal α-helix within GP-2 did not oligomerize. Refolding of the complete GP-2 ectodomain revealed trimeric all-alpha complexes probably representing the six-helix bundle state that is considered a hallmark of class I viral fusion proteins. Based on these results, we generated a construct consisting of the complete uncleavable LCMV GP ectodomain fused C-terminally to the trimeric motif of fibritin. Gel filtration analysis of the secreted fusion protein identified two complexes of ∼230 and ∼440 kDa. Both complexes bound to a set of conformational and linear antibodies. Cross-linking confirmed the 230-kDa complex to be a trimer. The 440-kDa complexes were found to represent disulfide-linked pairs of trimers, since partial reduction converted them to a complex species migrating at 250 kDa. By electron microscopy, the 230-kDa complexes appeared as single spherical particles and showed no signs of rosette formation. Our results clearly demonstrate that the arenavirus GP is a trimer and must be considered a member of the class I viral fusion protein family.

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