Virus Membrane Fusion Proteins: Biological Machines that Undergo a Metamorphosis

2000 ◽  
Vol 20 (6) ◽  
pp. 597-612 ◽  
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.

scholarly journals 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

2007 ◽  
Vol 82 (5) ◽  
pp. 2437-2447 ◽  
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.

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

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. e00047-18 ◽  
Author(s):  
Stacy R. Webb ◽  
Stacy E. Smith ◽  
Michael G. Fried ◽  
Rebecca Ellis Dutch
Keyword(s):  
Fusion Protein ◽  
Conformational Changes ◽  
Fusion Proteins ◽  
Ebola Virus ◽  
Transmembrane Domain ◽  
Transmembrane Domains ◽  
Viral Fusion ◽  
Viral Fusion Proteins ◽  
The Stability ◽  
The Right

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.

scholarly journals Sequential Roles of Receptor Binding and Low pH in Forming Prehairpin and Hairpin Conformations of a Retroviral Envelope Glycoprotein

2004 ◽  
Vol 78 (15) ◽  
pp. 8201-8209 ◽  
Author(s):  
Shutoku Matsuyama ◽  
Sue Ellen Delos ◽  
Judith M. White
Keyword(s):  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Fusion Peptide ◽  
Low Ph ◽  
Class I ◽  
Viral Fusion ◽  
Conformational States ◽  
Helix Bundle ◽  
Subsequent Exposure ◽  
Viral Fusion Proteins

ABSTRACT A general model has been proposed for the fusion mechanisms of class I viral fusion proteins. According to this model a metastable trimer, anchored in the viral membrane through its transmembrane domain, transits to a trimeric prehairpin intermediate, anchored at its opposite end in the target membrane through its fusion peptide. A subsequent refolding event creates a trimer of hairpins (often termed a six-helix bundle) in which the previously well-separated transmembrane domain and fusion peptide (and their attached membranes) are brought together, thereby driving membrane fusion. While there is ample biochemical and structural information on the trimer-of-hairpins conformation of class I viral fusion proteins, less is known about intermediate states between native metastable trimers and the final trimer of hairpins. In this study we analyzed conformational states of the transmembrane subunit (TM), the fusion subunit, of the Env glycoprotein of the subtype A avian sarcoma and leukosis virus (ASLV-A). By analyzing forms of EnvA TM on mildly denaturing sodium dodecyl sulfate gels we identified five conformational states of EnvA TM. Following interaction of virions with a soluble form of the ASLV-A receptor at 37°C, the metastable form of EnvA TM (which migrates at 37 kDa) transits to a 70-kDa and then to a 150-kDa species. Following subsequent exposure to a low pH (or an elevated temperature or the fusion promoting agent chlorpromazine), an additional set of bands at >150 kDa, and then a final band at 100 kDa, forms. Both an EnvA C-helix peptide (which inhibits virus fusion and infectivity) and the fusion-inhibitory agent lysophosphatidylcholine inhibit the formation of the >150- and 100-kDa bands. Our data are consistent with the 70- and 150-kDa bands representing precursor and fully formed prehairpin conformations of EnvA TM. Our data are also consistent with the >150-kDa bands representing higher-order oligomers of EnvA TM and with the 100-kDa band representing the fully formed six-helix bundle. In addition to resolving fusion-relevant conformational intermediates of EnvA TM, our data are compatible with a model in which the EnvA protein is activated by its receptor (at neutral pH and a temperature greater than or equal to room temperature) to form prehairpin conformations of EnvA TM, and in which subsequent exposure to a low pH is required to stabilize the final six-helix bundle, which drives a later stage of fusion.

scholarly journals Viral Membrane Fusion and the Transmembrane Domain

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 693 ◽  
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.

scholarly journals Mutations in the Fusion Peptide and Adjacent Heptad Repeat Inhibit Folding or Activity of the Newcastle Disease Virus Fusion Protein

2001 ◽  
Vol 75 (17) ◽  
pp. 7934-7943 ◽  
Author(s):  
Theresa A. Sergel ◽  
Lori W. McGinnes ◽  
Trudy G. Morrison
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Newcastle Disease Virus ◽  
Newcastle Disease ◽  
Coiled Coil ◽  
Surface Expression ◽  
Fusion Peptide ◽  
Proteolytic Cleavage ◽  
Heptad Repeat ◽  
Fusion Activity

ABSTRACT Paramyxovirus fusion proteins have two heptad repeat domains, HR1 and HR2, which have been implicated in the fusion activity of the protein. Peptides with sequences from these two domains form a six-stranded coiled coil, with the HR1 sequences forming a central trimer (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999; X. Zhao, M. Singh, V. N. Malashkevich, and P. S. Kim, Proc. Natl. Acad. Sci. USA 97:14172–14177, 2000). We have extended our previous mutational analysis of the HR1 domain of the Newcastle disease virus fusion protein, focusing on the role of the amino acids forming the hydrophobic core of the trimer, amino acids in the “a” and “d” positions of the helix from amino acids 123 to 182. Both conservative and nonconservative point mutations were characterized for their effects on synthesis, stability, proteolytic cleavage, and surface expression. Mutant proteins expressed on the cell surface were characterized for fusion activity by measuring syncytium formation, content mixing, and lipid mixing. We found that all mutations in the “a” position interfered with proteolytic cleavage and surface expression of the protein, implicating the HR1 domain in the folding of the F protein. However, mutation of five of seven “d” position residues had little or no effect on surface expression but, with one exception at residue 175, did interfere to various extents with the fusion activity of the protein. One of these “d” mutations, at position 154, interfered with proteolytic cleavage, while the rest of the mutants were cleaved normally. That most “d” position residues do affect fusion activity argues that a stable HR1 trimer is required for formation of the six-stranded coiled coil and, therefore, optimal fusion activity. That most of the “d” position mutations do not block folding suggests that formation of the core trimer may not be required for folding of the prefusion form of the protein. We also found that mutations within the fusion peptide, at residue 128, can interfere with folding of the protein, implicating this region in folding of the molecule. No characterized mutation enhanced fusion.

scholarly journals Cathepsin L Functionally Cleaves the Severe Acute Respiratory Syndrome Coronavirus Class I Fusion Protein Upstream of Rather than Adjacent to the Fusion Peptide

2008 ◽  
Vol 82 (17) ◽  
pp. 8887-8890 ◽  
Author(s):  
Berend Jan Bosch ◽  
Willem Bartelink ◽  
Peter J. M. Rottier
Keyword(s):  
Fusion Proteins ◽  
Cathepsin L ◽  
Fusion Peptide ◽  
Cell Entry ◽  
Spike Protein ◽  
Class I ◽  
Viral Fusion ◽  
Cathepsin Proteases ◽  
Viral Fusion Proteins ◽  
Binding Subunit

ABSTRACT Unlike other class I viral fusion proteins, spike proteins on severe acute respiratory sydrome coronavirus virions are uncleaved. As we and others have demonstrated, infection by this virus depends on cathepsin proteases present in endosomal compartments of the target cell, suggesting that the spike protein acquires its fusion competence by cleavage during cell entry rather than during virion biogenesis. Here we demonstrate that cathepsin L indeed activates the membrane fusion function of the spike protein. Moreover, cleavage was mapped to the same region where, in coronaviruses carrying furin-activated spikes, the receptor binding subunit of the protein is separated from the membrane-anchored fusion subunit.

scholarly journals MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, and Avian Influenza Virus H5N1 Hemagglutinin Maturation

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Changqing Yu ◽  
Sunan Li ◽  
Xianfeng Zhang ◽  
Ilyas Khan ◽  
Iqbal Ahmad ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusion Proteins ◽  
Ebola Virus ◽  
Viral Fusion ◽  
Virus H5n1 ◽  
Immunodeficiency Virus ◽  
Avian Influenza Virus H5n1 ◽  
Viral Fusion Proteins ◽  
Hiv 1

ABSTRACT Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions. IMPORTANCE Enveloped viruses express three classes of fusion proteins that are required for their entry into host cells via mediating virus and cell membrane fusion. Class I fusion proteins are produced from influenza viruses, retroviruses, Ebola viruses, and coronaviruses. They are first synthesized as a type I transmembrane polypeptide precursor that is subsequently glycosylated and oligomerized. Most of these precursors are cleaved en route to the plasma membrane by a cellular protease furin in the late secretory pathway, generating the trimeric N-terminal receptor-binding and C-terminal fusion subunits. Here, we show that a cellular protein, MARCH8, specifically inhibits the furin-mediated cleavage of EBOV GP, HIV-1 Env, and H5N1 HA. Further analyses uncovered that MARCH8 blocked the EBOV GP glycosylation in the Golgi and inhibited its transport from the Golgi to the plasma membrane. Thus, MARCH8 has a very broad antiviral activity by specifically inactivating different viral fusion proteins.

scholarly journals The Coronavirus Spike Protein Is a Class I Virus Fusion Protein: Structural and Functional Characterization of the Fusion Core Complex

2003 ◽  
Vol 77 (16) ◽  
pp. 8801-8811 ◽  
Author(s):  
Berend Jan Bosch ◽  
Ruurd van der Zee ◽  
Cornelis A. M. de Haan ◽  
Peter J. M. Rottier
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Limited Proteolysis ◽  
Fusion Peptide ◽  
Spike Protein ◽  
Class I ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Viral Fusion Protein

ABSTRACT Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure ∼14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.

Structural comparison of three viral “fusion” proteins

1991 ◽  
Vol 19 (3) ◽  
pp. 312S-312S
Author(s):  
BRUCE H NICHOLSON ◽  
MAHMOUD NAASE
Keyword(s):  
Fusion Proteins ◽  
Viral Fusion ◽  
Structural Comparison ◽  
Viral Fusion Proteins
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