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

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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Class II fusion protein of alphaviruses drives membrane fusion through the same pathway as class I proteins

The Journal of Cell Biology ◽

10.1083/jcb.200412059 ◽

2005 ◽

Vol 169 (1) ◽

pp. 167-177 ◽

Cited By ~ 50

Author(s):

Elena Zaitseva ◽

Aditya Mittal ◽

Diane E. Griffin ◽

Leonid V. Chernomordik

Keyword(s):

Influenza Virus ◽

Fusion Proteins ◽

Low Ph ◽

Viral Fusion ◽

Fusion Reactions ◽

Influenza Virus Hemagglutinin ◽

Viral Fusion Proteins ◽

The Difference ◽

Fusion Pores ◽

Specific Inhibitors

Viral fusion proteins of classes I and II differ radically in their initial structures but refold toward similar conformations upon activation. Do fusion pathways mediated by alphavirus E1 and influenza virus hemagglutinin (HA) that exemplify classes II and I differ to reflect the difference in their initial conformations, or concur to reflect the similarity in the final conformations? Here, we dissected the pathway of low pH–triggered E1-mediated cell–cell fusion by reducing the numbers of activated E1 proteins and by blocking different fusion stages with specific inhibitors. The discovered progression from transient hemifusion to small, and then expanding, fusion pores upon an increase in the number of activated fusion proteins parallels that established for HA-mediated fusion. We conclude that proteins as different as E1 and HA drive fusion through strikingly similar membrane intermediates, with the most energy-intensive stages following rather than preceding hemifusion. We propose that fusion reactions catalyzed by all proteins of both classes follow a similar pathway.

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Role of Electrostatic Repulsion in Controlling pH-Dependent Conformational Changes of Viral Fusion Proteins

Structure ◽

10.1016/j.str.2013.05.009 ◽

2013 ◽

Vol 21 (7) ◽

pp. 1085-1096 ◽

Cited By ~ 34

Author(s):

Joseph S. Harrison ◽

Chelsea D. Higgins ◽

Matthew J. O’Meara ◽

Jayne F. Koellhoffer ◽

Brian A. Kuhlman ◽

...

Keyword(s):

Conformational Changes ◽

Fusion Proteins ◽

Electrostatic Repulsion ◽

Viral Fusion ◽

Viral Fusion Proteins ◽

Ph Dependent

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A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion

Molecular Biology of the Cell ◽

10.1091/mbc.10.12.4191 ◽

1999 ◽

Vol 10 (12) ◽

pp. 4191-4200 ◽

Cited By ~ 38

Author(s):

David H. Kingsley ◽

Ali Behbahani ◽

Afshin Rashtian ◽

Gary W. Blissard ◽

Joshua Zimmerberg

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Conformational Changes ◽

Fusion Proteins ◽

Critical Role ◽

Low Ph ◽

Heptad Repeat ◽

Viral Fusion ◽

Viral Fusion Protein ◽

Heptad Repeats

Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH–induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.

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Histidine protonation and the activation of viral fusion proteins

Biochemical Society Transactions ◽

10.1042/bst0360043 ◽

2008 ◽

Vol 36 (1) ◽

pp. 43-45 ◽

Cited By ~ 46

Author(s):

Daniela S. Mueller ◽

Thorsten Kampmann ◽

Ragothaman Yennamalli ◽

Paul R. Young ◽

Bostjan Kobe ◽

...

Keyword(s):

Conformational Changes ◽

Envelope Protein ◽

Fusion Proteins ◽

Side Chain ◽

Viral Fusion ◽

Histidine Residues ◽

Local Environments ◽

Viral Fusion Proteins ◽

Dynamics Simulations

Many viral fusion proteins only become activated under mildly acidic condition (pH 4.5–6.5) close to the pKa of histidine side-chain protonation. Analysis of the sequences and structures of influenza HA (haemagglutinin) and flaviviral envelope glycoproteins has led to the identification of a number of histidine residues that are not only fully conserved themselves but have local environments that are also highly conserved [Kampmann, Mueller, Mark, Young and Kobe (2006) Structure 14, 1481–1487]. Here, we summarize studies aimed at determining the role, if any, that protonation of these potential switch histidine residues plays in the low-pH-dependent conformational changes associated with fusion activation of a flaviviral envelope protein. Specifically, we report on MD (Molecular Dynamics) simulations of the DEN2 (dengue virus type 2) envelope protein ectodomain sE (soluble E) performed under varied pH conditions designed to test the histidine switch hypothesis of Kampmann et al. (2006).

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Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618883114 ◽

2016 ◽

Vol 114 (1) ◽

pp. E28-E36 ◽

Cited By ~ 49

Author(s):

Irene S. Kim ◽

Simon Jenni ◽

Megan L. Stanifer ◽

Eatai Roth ◽

Sean P. J. Whelan ◽

...

Keyword(s):

Membrane Fusion ◽

G Protein ◽

Vesicular Stomatitis Virus ◽

Fusion Proteins ◽

Low Ph ◽

Viral Fusion ◽

Protein Fusion ◽

West Nile ◽

Vesicular Stomatitis ◽

Viral Fusion Proteins

The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a “class I” fusogen) and West Nile virus envelope protein (“class II”). Our study of VSV now extends this description to “class III” viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion.

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

mSphere ◽

10.1128/msphere.00047-18 ◽

2018 ◽

Vol 3 (2) ◽

pp. e00047-18 ◽

Cited By ~ 6

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.

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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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