Peptide-based Fusion Inhibitors for Preventing the Six-helix Bundle Formation of Class I Fusion Proteins: HIV and Beyond

: A number of different viral families have developed convergent methods to infect cells. Class I fusion proteins are commonly used by members of Arenaviridae, Coronaviridae, Filovirdae, Orthomyxoviridae, Paramyxoviridae, and Retroviridae. Class I viral fusion proteins are trimers that are involved in recognizing the cellular receptor, with a region that is responsible for fusing the viral and target cell membranes. During the fusion process, the fusion region folds into a six-helix bundle (6HB) which approximates the two membranes leading to fusion. For human immunodeficiency virus (HIV), the gp41 subunit is responsible for the formation of this 6HB. The fusion inhibitor drug enfuvirtide, or T20, is the only US Food and Drug Administration and European Medicines Agency approved drug which targets this crucial step and has been widely used in combination regimens for the treatment of HIV since March 2003. In this review, we describe the current state of peptide-based fusion inhibitors in the treatment of HIV, and review how the field of HIV research is driving advances in the development of similar therapeutics in other viral systems, including the severe acute respiratory syndrome (SARS) coronaviruses.

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

Journal of Virology ◽

10.1128/jvi.78.15.8201-8209.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8201-8209 ◽

Cited By ~ 50

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.

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Characterization of EBV gB indicates properties of both class I and class II viral fusion proteins

Virology ◽

10.1016/j.virol.2007.06.031 ◽

2007 ◽

Vol 368 (1) ◽

pp. 102-113 ◽

Cited By ~ 24

Author(s):

Marija Backovic ◽

George P. Leser ◽

Robert A. Lamb ◽

Richard Longnecker ◽

Theodore S. Jardetzky

Keyword(s):

Fusion Proteins ◽

Class Ii ◽

Class I ◽

Viral Fusion ◽

Viral Fusion Proteins

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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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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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Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins

Journal of Virology ◽

10.1128/jvi.00015-16 ◽

2016 ◽

Vol 90 (12) ◽

pp. 5586-5600 ◽

Cited By ~ 30

Author(s):

Xiuyuan Ou ◽

Wangliang Zheng ◽

Yiwei Shan ◽

Zhixia Mu ◽

Samuel R. Dominguez ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Cell Fusion ◽

Fusion Proteins ◽

Virus Entry ◽

Amino Acid Sequences ◽

Viral Envelope ◽

Class I ◽

Viral Fusion ◽

Viral Fusion Proteins

ABSTRACTThe fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide (955IAGVGWTAGL964) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other β-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of β-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution.IMPORTANCEWithin the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 β-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of β-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of β-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.

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

Journal of Virology ◽

10.1128/jvi.00415-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8887-8890 ◽

Cited By ~ 144

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.

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

Journal of Virology ◽

10.1128/jvi.77.16.8801-8811.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 8801-8811 ◽

Cited By ~ 605

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.

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