Hemagglutinin-Mediated Membrane Fusion: A Biophysical Perspective

Influenza hemagglutinin (HA) is a viral membrane protein responsible for the initial steps of the entry of influenza virus into the host cell. It mediates binding of the virus particle to the host-cell membrane and catalyzes fusion of the viral membrane with that of the host. HA is therefore a major target in the development of antiviral strategies. The fusion of two membranes involves high activation barriers and proceeds through several intermediate states. Here, we provide a biophysical description of the membrane fusion process, relating its kinetic and thermodynamic properties to the large conformational changes taking place in HA and placing these in the context of multiple HA proteins working together to mediate fusion. Furthermore, we highlight the role of novel single-particle experiments and computational approaches in understanding the fusion process and their complementarity with other biophysical approaches.

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Structural intermediates in the low pH-induced transition of influenza hemagglutinin

PLoS Pathogens ◽

10.1371/journal.ppat.1009062 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1009062

Author(s):

Jingjing Gao ◽

Miao Gui ◽

Ye Xiang

Keyword(s):

Host Cell ◽

Conformational Change ◽

Conformational Changes ◽

Virus Entry ◽

Low Ph ◽

Influenza Viruses ◽

Fusion Peptides ◽

Host Cell Membrane ◽

Influenza Hemagglutinin ◽

Binding Pockets

The hemagglutinin (HA) glycoproteins of influenza viruses play a key role in binding host cell receptors and in mediating virus-host cell membrane fusion during virus infection. Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. While structures of prefusion state and postfusion state of HA have been reported, the intermediate structures remain elusive. Here, we report two distinct low pH intermediate conformations of the influenza virus HA using cryo-electron microscopy (cryo-EM). Our results show that a decrease in pH from 7.8 to 5.2 triggers the release of fusion peptides from the binding pockets and then causes a dramatic conformational change in the central helices, in which the membrane-proximal ends of the central helices unwind to an extended form. Accompanying the conformational changes of the central helices, the stem region of the HA undergoes an anticlockwise rotation of 9.5 degrees and a shift of 15 Å. The HA head, after being stabilized by an antibody, remains unchanged compared to the neutral pH state. Thus, the conformational change of the HA stem region observed in our research is likely to be independent of the HA head. These results provide new insights into the structural transition of HA during virus entry.

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Insights into a Structure-Based Mechanism of Viral Membrane Fusion

Bioscience Reports ◽

10.1023/a:1010463005396 ◽

2000 ◽

Vol 20 (6) ◽

pp. 557-570 ◽

Cited By ~ 9

Author(s):

Danika L. LeDuc ◽

Yeon-Kyun Shin

Keyword(s):

Membrane Fusion ◽

General Structure ◽

Viral Membrane ◽

Induced Membrane ◽

Alternative Scenario ◽

Influenza Hemagglutinin ◽

Hiv Gp120 ◽

Viral Membrane Fusion ◽

Spike Proteins

A number of different viral spike proteins, responsible for membrane fusion, show striking similarities in their core structures. The prospect of developing a general structure-based mechanism seems plausible in light of these newly determined structures. Influenza hemagglutinin (HA) is the best-studied fusion machine, whose action has previously been described by a hypothetical “spring-loaded” model. This model has recently been extended to explain the mechanism of other systems, such as HIV gp120–gp41. However, evidence supporting this idea is insufficient, requiring re-examination of the mechanism of HA-induced membrane fusion. Recent experiments with a shortened construct of HA, which is able to induce lipid mixing, have provided evidence for an alternative scenario for HA-induced membrane fusion and perhaps that of other viral systems.

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The Role of Histidine Residues in Low-pH-Mediated Viral Membrane Fusion

Structure ◽

10.1016/j.str.2006.07.011 ◽

2006 ◽

Vol 14 (10) ◽

pp. 1481-1487 ◽

Cited By ~ 106

Author(s):

Thorsten Kampmann ◽

Daniela S. Mueller ◽

Alan E. Mark ◽

Paul R. Young ◽

Bostjan Kobe

Keyword(s):

Membrane Fusion ◽

Low Ph ◽

Viral Membrane ◽

Histidine Residues ◽

Viral Membrane Fusion

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An Investigation of the Influenza Hemagglutinin Membrane Fusion Process using Microsecond-Level MD Simulations

Biophysical Journal ◽

10.1016/j.bpj.2019.11.491 ◽

2020 ◽

Vol 118 (3) ◽

pp. 57a

Author(s):

Vivek Govind Kumar ◽

Dylan S. Ogden ◽

Adithya Polasa ◽

Mahmoud Moradi

Keyword(s):

Membrane Fusion ◽

Md Simulations ◽

Fusion Process ◽

Influenza Hemagglutinin

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Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion

Journal of Virology ◽

10.1128/jvi.01350-20 ◽

2020 ◽

Vol 94 (24) ◽

Author(s):

Angke Zhang ◽

Hong Duan ◽

Huijun Zhao ◽

Huancheng Liao ◽

Yongkun Du ◽

...

Keyword(s):

Membrane Fusion ◽

Transmembrane Protein ◽

Fluorescent Labeling ◽

Host Cells ◽

Respiratory Syndrome Virus ◽

Type I ◽

Antiviral Immune Response ◽

Viral Membrane ◽

Viral Membrane Fusion

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) infection eliminates production of type I interferons (IFNs) in host cells, which triggers an antiviral immune response through the induction of downstream IFN-stimulated genes (ISGs), thus escaping the fate of host-mediated clearance. The IFN-induced transmembrane 3 (IFITM3) has recently been identified as an ISG and plays a pivotal role against enveloped RNA viruses by restricting cell entry. However, the role of IFITM3 in PRRSV replication is unknown. The present study demonstrated that overexpression of IFITM3 suppresses PRRSV replication, while silencing of endogenous IFITM3 prominently promoted PRRSV replication. Additionally, it was shown that IFITM3 undergoes S-palmitoylation and ubiquitination modification, and both posttranslational modifications contribute to the anti-PRRSV activity of IFITM3. Further study showed that PRRSV particles are transported into endosomes and then into lysosomes during the early stages of infection, and confocal microscopy results revealed that PRRSV particles are transported to IFITM3-positive cellular vesicles. By using a single virus particle fluorescent labeling technique, we confirmed that IFITM3 can restrict PRRSV membrane fusion by inducing accumulation of cholesterol in cellular vesicles. Additionally, we found that both endogenous and exogenous IFITM3 are incorporated into newly producing PRRS virions and diminish viral intrinsic infectivity. By using cell coculture systems, we found that IFITM3 effectively restricted PRRSV intercellular transmission, which may have been caused by disrupted membrane fusion and reduced viral infectivity. In conclusion, our results demonstrate, for the first time, that swine IFITM3 interferes with the life cycle of PRRSV, and possibly other enveloped arteritis viruses, at multiple steps. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is of great economic significance to the swine industry. Due to the complicated immune escape mechanisms of PRRSV, there are no effective vaccines or therapeutic drugs currently available against PRRS. Identification of cellular factors and underlying mechanisms that establish an effective antiviral state against PRRSV can provide unique strategies for developing antiviral vaccines or drugs. As an interferon (IFN)-stimulated gene, the role of IFN-induced transmembrane 3 (IFITM3) in PRRSV infection has not been reported as of yet. In the present study, it was shown that IFITM3 can exert a potent anti-PRRSV effect, and PRRS virions are trafficked to IFITM3-containing cell vesicles, where viral membrane fusion is impaired by cholesterol accumulation that is induced by IFITM3. Additionally, both endogenous and exogenous IFITM3 are incorporated into newly assembled progeny virions, and this decreased their intrinsic infectivity.

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Third Helical Domain of the Nipah Virus Fusion Glycoprotein Modulates both Early and Late Steps in the Membrane Fusion Cascade

Journal of Virology ◽

10.1128/jvi.00644-20 ◽

2020 ◽

Vol 94 (19) ◽

Author(s):

J. Lizbeth Reyes Zamora ◽

Victoria Ortega ◽

Gunner P. Johnston ◽

Jenny Li ◽

Nicole M. André ◽

...

Keyword(s):

Membrane Fusion ◽

Host Range ◽

Viral Entry ◽

Nipah Virus ◽

Fusion Process ◽

Fusion Pore ◽

Animal Pathogens ◽

Fusion Glycoprotein ◽

Helical Region

ABSTRACT Medically important paramyxoviruses, such as measles, mumps, parainfluenza, Nipah, and Hendra viruses, infect host cells by directing fusion of the viral and cellular plasma membranes. Upon infection, paramyxoviruses cause a second type of membrane fusion, cell-cell fusion (syncytium formation), which is linked to pathogenicity. Host cell receptor binding causes conformational changes in the attachment glycoprotein (HN, H, or G) that trigger a conformational cascade in the fusion (F) glycoprotein that mediates membrane fusion. F, a class I fusion protein, contains the archetypal heptad repeat regions 1 (HR1) and 2 (HR2). It is well established that binding of HR1 and HR2 is key to fusing viral and cellular membranes. In this study, we uncovered a novel fusion-modulatory role of a third structurally conserved helical region (HR3) in F. Based on its location within the F structure, and structural differences between its prefusion and postfusion conformations, we hypothesized that the HR3 modulates triggering of the F conformational cascade (still requiring G). We used the deadly Nipah virus (NiV) as an important paramyxoviral model to perform alanine scan mutagenesis and a series of multidisciplinary structural/functional analyses that dissect the various states of the membrane fusion cascade. Remarkably, we found that specific residues within the HR3 modulate not only early F-triggering but also late extensive fusion pore expansion steps in the membrane fusion cascade. Our results characterize these novel fusion-modulatory roles of the F HR3, improving our understanding of the membrane fusion process for NiV and likely for the related Henipavirus genus and possibly Paramyxoviridae family members. IMPORTANCE The Paramyxoviridae family includes important human and animal pathogens, such as measles, mumps, and parainfluenza viruses and the deadly henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviruses infect the respiratory tract and the central nervous system (CNS) and can be highly infectious. Most paramyxoviruses have a limited host range. However, the biosafety level 4 NiV and HeV are highly pathogenic and have a wide mammalian host range. Nipah viral infections result in acute respiratory syndrome and severe encephalitis in humans, leading to 40 to 100% mortality rates. The lack of licensed vaccines or therapeutic approaches against NiV and other important paramyxoviruses underscores the need to understand viral entry mechanisms. In this study, we uncovered a novel role of a third helical region (HR3) of the NiV fusion glycoprotein in the membrane fusion process that leads to viral entry. This discovery sets HR3 as a new candidate target for antiviral strategies for NiV and likely for related viruses.

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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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Role of the Hemagglutinin-Neuraminidase Protein in the Mechanism of Paramyxovirus-Cell Membrane Fusion

Journal of Virology ◽

10.1128/jvi.76.24.13028-13033.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 13028-13033 ◽

Cited By ~ 114

Author(s):

Toru Takimoto ◽

Garry L. Taylor ◽

Helen C. Connaris ◽

Susan J. Crennell ◽

Allen Portner

Keyword(s):

Plasma Membrane ◽

Membrane Fusion ◽

Conformational Changes ◽

Hydrophobic Surface ◽

Cellular Receptor ◽

Virus Attachment ◽

Acetylneuraminic Acid ◽

Specific Manner ◽

Β Sheet

ABSTRACT Paramyxovirus infects cells by initially attaching to a sialic acid-containing cellular receptor and subsequently fusing with the plasma membrane of the cells. Hemagglutinin-neuraminidase (HN) protein, which is responsible for virus attachment, interacts with the fusion protein in a virus type-specific manner to induce efficient membrane fusion. To elucidate the mechanism of HN-promoted membrane fusion, we characterized a series of Newcastle disease virus HN proteins whose surface residues were mutated. Fusion promotion activity was substantially altered in only the HN proteins with a mutation in the first or sixth β sheet. These regions overlap the large hydrophobic surface of HN; thus, the hydrophobic surface may contain the fusion promotion domain. Furthermore, a comparison of the HN structure crystallized alone or in complex with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid revealed substantial conformational changes in several loops within or near the hydrophobic surface. Our results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of HN protein and that this change triggers the activation of the F protein, which initiates membrane fusion.

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Characterization of Fusion Determinants Points to the Involvement of Three Discrete Regions of Both E1 and E2 Glycoproteins in the Membrane Fusion Process of Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.02642-06 ◽

2007 ◽

Vol 81 (16) ◽

pp. 8752-8765 ◽

Cited By ~ 118

Author(s):

Dimitri Lavillette ◽

Eve-Isabelle Pécheur ◽

Peggy Donot ◽

Judith Fresquet ◽

Jennifer Molle ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Membrane Fusion ◽

Conformational Changes ◽

Lipid Membranes ◽

Fusion Process ◽

Fusion Peptides ◽

Cellular Membranes ◽

Enveloped Virus ◽

Molecular Events

ABSTRACT Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.

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pH-induced alterations in the fusogenic spike protein of Semliki Forest virus.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2284 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2284-2291 ◽

Cited By ~ 110

Author(s):

M Kielian ◽

A Helenius

Keyword(s):

Membrane Fusion ◽

Conformational Changes ◽

Semliki Forest Virus ◽

Fusion Reaction ◽

Viral Envelope ◽

Acidic Ph ◽

Binding Assays ◽

Protease Digestion ◽

Nonionic Detergent

The spike glycoproteins of Semliki Forest virus mediate membrane fusion between the viral envelope and cholesterol-containing target membranes under conditions of mildly acidic pH (pH less than 6.2). The fusion reaction is critical for the infectious cycle, catalyzing virus penetration from the acidic endosome compartment. To define the role of the viral spike glycoproteins in the fusion reaction, conformational changes in the spikes at acid pH were studied using protease digestion and binding assays to liposomes and nonionic detergent. A method was also developed to prepare fragments of both transmembrane subunit glycopolypeptides of the spike, E1 and E2, which lacked the hydrophobic anchor peptides. Unlike the intact spikes the fragments were monomeric and therefore useful for obtaining information on conformational changes in individual subunits. The results showed that both E1 and E2 undergo irreversible conformational changes at the pH of fusion, that the conformational change of E1 depends, in addition to acidic pH, on the presence of cholesterol, and that no major changes in the solubility properties of the spikes takes place. On the basis of these findings it was concluded that fusion involves both subunits of the spike and that E1 confers the stereo-specific sterol requirement. The results indicated, moreover, that acid-induced fusion of Semliki Forest virus differs in important respects from that of influenza virus, another well-defined model system for protein-mediated membrane fusion.

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