Special Issue "Mechanisms of Viral Fusion and Applications in Antivirals"

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Antivirals & Vaccines".

Deadline for manuscript submissions: closed (31 December 2019).

Special Issue Editors

Dr. Sergi Padilla-Parra
Website
Guest Editor
Head of Core in Cellular Imaging and Principal Investigator at Division of Structural Biology, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford, OX3 7BN, UK
Interests: mechanism of HIV-1 virus fusion employing single molecule approaches
Prof. Dr. Shan-Lu Liu
Website
Guest Editor
Director, Viruses and Emerging Pathogens Program, Infectious Diseases Institute, The Ohio State University;
Professor, Center for Retrovirus Research, Department of Veterinary Biosciences, Department of Microbial Infection and Immunity, The Ohio State University, 1900 Coffey Rd., Columbus, OH 43210, USA
Interests: viral membrane fusion and entry; cellular restriction factors against viral infection; retroviral oncogenesis; viral envelope glycoproteins; lentiviral vectors and gene therapy

Special Issue Information

Dear colleague,

Virus-cell fusion is essential for enveloped viruses to enter host cells. Enveloped viruses acquire their membrane from infected host cells during the budding process. Many important human pathogens are enveloped viruses, such as human immunodeficiency virus (HIV), Hepatitis C (HCV), and Ebola virus (EBOV). The virus–cell fusion process is carried out by one or more viral envelope glycoprotein or fusion proteins. Viral fusion proteins contain a typical fusion peptide or a fusion loop that is responsible for initial insertion into host membrane, and they are normally present in virions as a prefusion state, which must be triggered to mediate fusion and viral entry. The current known triggers include receptor binding, low pH, receptor-binding plus low pH, and cellular cathepsins, but may also include calcium and certain lipids. However, regardless of distinct prefusion structures and triggers, the post-fusion state of all viral fusion proteins is a trimer of hairpin.

In this Special Issue, we will focus on the most recent advances in understanding the mechanism of virus–cell fusion, with a special emphasis on advanced imaging approaches (single molecule techniques and super-resolution) and viral fusion triggers. We will also focus on new developments on neutralizing antibodies, antivirals, and vaccine development.

Dr. Sergi Padilla-Parra
Prof. Shan-Lu Liu
Guest Editors

Manuscript Submission Information

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Keywords

  • virus–cell fusion mechanism 
  • fusion triggers 
  • single viral particle imaging 
  • membrane fusion kinetics
  • advanced microscopy
  • neutralizing antibodies 
  • small molecule fusion inhibitors 
  • antiviral drugs

Published Papers (13 papers)

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Research

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Open AccessArticle
Identification of an Intermediate Step in Foamy Virus Fusion
Viruses 2020, 12(12), 1472; https://doi.org/10.3390/v12121472 - 21 Dec 2020
Abstract
Viral glycoprotein-mediated membrane fusion is an essential step for productive infection of host cells by enveloped viruses; however, due to its rarity and challenges in detection, little is known about the details of fusion events at the single particle level. Here, we have [...] Read more.
Viral glycoprotein-mediated membrane fusion is an essential step for productive infection of host cells by enveloped viruses; however, due to its rarity and challenges in detection, little is known about the details of fusion events at the single particle level. Here, we have developed dual-color foamy viruses (FVs) composed of eGFP-tagged prototype FV (PFV) Gag and mCherry-tagged Env of either PFV or macaque simian FV (SFVmac) origin that have been optimized for detection of the fusion process. Using our recently developed tracking imaging correlation (TrIC) analysis, we were able to detect the fusion process for both PFV and SFVmac Env containing virions. PFV Env-mediated fusion was observed both at the plasma membrane as well as from endosomes, whereas SFVmac Env-mediated fusion was only observed from endosomes. PFV Env-mediated fusion was observed to happen more often and more rapidly than as for SFVmac Env. Strikingly, using the TrIC method, we detected a novel intermediate state where the envelope and capsids are still tethered but separated by up to 400 nm before final separation of Env and Gag occurred. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessArticle
The Mutation of the Genes Related to Neurovirulence in HSV-2 Produces an Attenuated Phenotype in Mice
Viruses 2020, 12(7), 770; https://doi.org/10.3390/v12070770 - 17 Jul 2020
Abstract
HSV-2 (Herpes simplex virus type 2) is a critical viral agent that mainly causes genital herpes and life-long latent infection in the dorsal root ganglia. Gene modification via CRISPR/Cas9 Clustered regularly interspaced short palindromic repeat sequences/CRISPR associated 9) was used here to construct [...] Read more.
HSV-2 (Herpes simplex virus type 2) is a critical viral agent that mainly causes genital herpes and life-long latent infection in the dorsal root ganglia. Gene modification via CRISPR/Cas9 Clustered regularly interspaced short palindromic repeat sequences/CRISPR associated 9) was used here to construct HSV-2 mutant strains through the deletion of fragments of the RL1 (Repeat Long element 1) and/or LAT (Latency-associated Transcript) genes. The HSV-2 mutant strains LAT-HSV-2 and RL1-LAT-HSV-2 present different biological properties. The proliferation of RL1-LAT-HSV-2 in nerve cells was decreased significantly, and the plaques induced by RL1-LAT-HSV-2 in Vero cells were smaller than those induced by LAT-HSV-2 mutant and wild-type strains. The observation of mice infected with these two mutants compared to mice infected with the wild-type strain indicated that the mutant RL1-LAT-HSV-2 has an attenuated phenotype with reduced pathogenicity during both acute and latent infections and induces a stronger specific immune response than the wild-type strain, whereas the attenuation effect was not found in mice infected with the LAT-HSV-2 mutant containing the LAT gene deletion. However, the simultaneous mutation of both the RL1 and LAT genes did not completely restrict viral proliferation in nerve cells, indicating that multiple HSV genes are involved in viral replication in the neural system. This work suggests that the HSV-2 genes RL1 and/or LAT might be involved in the virulence mechanisms in mouse infections. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessArticle
Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors
Viruses 2020, 12(3), 326; https://doi.org/10.3390/v12030326 - 18 Mar 2020
Cited by 1
Abstract
Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously [...] Read more.
Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessArticle
Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells
Viruses 2020, 12(2), 206; https://doi.org/10.3390/v12020206 - 12 Feb 2020
Abstract
The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to better define [...] Read more.
The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to better define both the molecular mechanistic underpinnings of this process but also the point of fusion and its kinetics. Here, we have developed a new method able to detect and quantify HIV-1 fusion in single live cells. We present a new approach that employs fluorescence lifetime imaging microscopy (FLIM) to detect Förster resonance energy transfer (FRET) when using the β-lactamase (BlaM) assay. This novel approach allows comparing different populations of single cells regardless the concentration of CCF2-AM FRET reporter in each cell, and more importantly, is able to determine the relative amount of viruses internalized per cell. We have applied this approach in both reporter TZM-bl cells and primary T cell lymphocytes. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessArticle
Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics
Viruses 2020, 12(1), 103; https://doi.org/10.3390/v12010103 - 15 Jan 2020
Cited by 4
Abstract
The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential [...] Read more.
The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessArticle
EWI-2 Inhibits Cell–Cell Fusion at the HIV-1 Virological Presynapse
Viruses 2019, 11(12), 1082; https://doi.org/10.3390/v11121082 - 20 Nov 2019
Cited by 1
Abstract
Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly efficient mode of HIV-1 transmission. While cell–cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, [...] Read more.
Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly efficient mode of HIV-1 transmission. While cell–cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, this is strongly inhibited by both viral (Gag) and host (ezrin and tetraspanins) proteins. Here, we identify EWI-2, a protein that was previously shown to associate with ezrin and tetraspanins, as a host factor that contributes to the inhibition of Env-mediated cell–cell fusion. Using quantitative fluorescence microscopy, shRNA knockdowns, and cell–cell fusion assays, we show that EWI-2 accumulates at the presynaptic terminal (i.e., the producer cell side of the VS), where it contributes to the fusion-preventing activities of the other viral and cellular components. We also find that EWI-2, like tetraspanins, is downregulated upon HIV-1 infection, most likely by Vpu. Despite the strong inhibition of fusion at the VS, T cell-based syncytia do form in vivo and in physiologically relevant culture systems, but they remain small. In regard to that, we demonstrate that EWI-2 and CD81 levels are restored on the surface of syncytia, where they (presumably) continue to act as fusion inhibitors. This study documents a new role for EWI-2 as an inhibitor of HIV-1-induced cell–cell fusion and provides novel insight into how syncytia are prevented from fusing indefinitely. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Review

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Open AccessReview
New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes
Viruses 2020, 12(4), 413; https://doi.org/10.3390/v12040413 - 08 Apr 2020
Cited by 3
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessReview
Structure-Based Design of Antivirals against Envelope Glycoprotein of Dengue Virus
Viruses 2020, 12(4), 367; https://doi.org/10.3390/v12040367 - 26 Mar 2020
Cited by 2
Abstract
Dengue virus (DENV) presents a significant threat to global public health with more than 500,000 hospitalizations and 25,000 deaths annually. Currently, there is no clinically approved antiviral drug to treat DENV infection. The envelope (E) glycoprotein of DENV is a promising target for [...] Read more.
Dengue virus (DENV) presents a significant threat to global public health with more than 500,000 hospitalizations and 25,000 deaths annually. Currently, there is no clinically approved antiviral drug to treat DENV infection. The envelope (E) glycoprotein of DENV is a promising target for drug discovery as the E protein is important for viral attachment and fusion. Understanding the structure and function of DENV E protein has led to the exploration of structure-based drug discovery of antiviral compounds and peptides against DENV infections. This review summarizes the structural information of the DENV E protein with regards to DENV attachment and fusion. The information enables the development of antiviral agents through structure-based approaches. In addition, this review compares the potency of antivirals targeting the E protein with the antivirals targeting DENV multifunctional enzymes, repurposed drugs and clinically approved antiviral drugs. None of the current DENV antiviral candidates possess potency similar to the approved antiviral drugs which indicates that more efforts and resources must be invested before an effective DENV drug materializes. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessReview
Fluorescence Microscopy of the HIV-1 Envelope
Viruses 2020, 12(3), 348; https://doi.org/10.3390/v12030348 - 21 Mar 2020
Cited by 2
Abstract
Human immunodeficiency virus (HIV) infection constitutes a major health and social issue worldwide. HIV infects cells by fusing its envelope with the target cell plasma membrane. This process is mediated by the viral Env glycoprotein and depends on the envelope lipid composition. Fluorescent [...] Read more.
Human immunodeficiency virus (HIV) infection constitutes a major health and social issue worldwide. HIV infects cells by fusing its envelope with the target cell plasma membrane. This process is mediated by the viral Env glycoprotein and depends on the envelope lipid composition. Fluorescent microscopy has been employed to investigate the envelope properties, and the processes of viral assembly and fusion, but the application of this technique to the study of HIV is still limited by a number of factors, such as the small size of HIV virions or the difficulty to label the envelope components. Here, we review fluorescence imaging studies of the envelope lipids and proteins, focusing on labelling strategies and model systems. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessReview
Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition
Viruses 2020, 12(3), 342; https://doi.org/10.3390/v12030342 - 20 Mar 2020
Cited by 1
Abstract
Paramyxoviruses and pneumoviruses infect cells through fusion (F) protein-mediated merger of the viral envelope with target membranes. Members of these families include a range of major human and animal pathogens, such as respiratory syncytial virus (RSV), measles virus (MeV), human parainfluenza viruses (HPIVs), [...] Read more.
Paramyxoviruses and pneumoviruses infect cells through fusion (F) protein-mediated merger of the viral envelope with target membranes. Members of these families include a range of major human and animal pathogens, such as respiratory syncytial virus (RSV), measles virus (MeV), human parainfluenza viruses (HPIVs), and highly pathogenic Nipah virus (NiV). High-resolution F protein structures in both the metastable pre- and the postfusion conformation have been solved for several members of the families and a number of F-targeting entry inhibitors have progressed to advanced development or clinical testing. However, small-molecule RSV entry inhibitors have overall disappointed in clinical trials and viral resistance developed rapidly in experimental settings and patients, raising the question of whether the available structural information may provide a path to counteract viral escape through proactive inhibitor engineering. This article will summarize current mechanistic insight into F-mediated membrane fusion and examine the contribution of structural information to the development of small-molecule F inhibitors. Implications are outlined for future drug target selection and rational drug engineering strategies. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessReview
Purinergic Receptors: Elucidating the Role of these Immune Mediators in HIV-1 Fusion
Viruses 2020, 12(3), 290; https://doi.org/10.3390/v12030290 - 07 Mar 2020
Abstract
Purinergic receptors are inflammatory mediators activated by extracellular nucleotides released by dying or injured cells. Several studies have described an important role for these receptors in HIV-1 entry, particularly regarding their activity on HIV-1 viral membrane fusion. Several reports identify purinergic receptor antagonists [...] Read more.
Purinergic receptors are inflammatory mediators activated by extracellular nucleotides released by dying or injured cells. Several studies have described an important role for these receptors in HIV-1 entry, particularly regarding their activity on HIV-1 viral membrane fusion. Several reports identify purinergic receptor antagonists that inhibit HIV-1 membrane fusion; these drugs are suspected to act through antagonizing Env-chemokine receptor interactions. They also appear to abrogate activity of downstream mediators that potentiate activation of the NLRP3 inflammasome pathway. Here we review the literature on purinergic receptors, the drugs that inhibit their function, and the evidence implicating these receptors in HIV-1 entry. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
Open AccessReview
Relating GPI-Anchored Ly6 Proteins uPAR and CD59 to Viral Infection
Viruses 2019, 11(11), 1060; https://doi.org/10.3390/v11111060 - 14 Nov 2019
Cited by 1
Abstract
The Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily protein is a group of molecules that share limited sequence homology but conserved three-fingered structures. Despite diverse cellular functions, such as in regulating host immunity, cell adhesion, and migration, the physiological roles of these [...] Read more.
The Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily protein is a group of molecules that share limited sequence homology but conserved three-fingered structures. Despite diverse cellular functions, such as in regulating host immunity, cell adhesion, and migration, the physiological roles of these factors in vivo remain poorly characterized. Notably, increasing research has focused on the interplays between Ly6/uPAR proteins and viral pathogens, the results of which have provided new insight into viral entry and virus–host interactions. While LY6E (lymphocyte antigen 6 family member E), one key member of the Ly6E/uPAR-family proteins, has been extensively studied, other members have not been well characterized. Here, we summarize current knowledge of Ly6/uPAR proteins related to viral infection, with a focus on uPAR and CD59. Our goal is to provide an up-to-date view of the Ly6/uPAR-family proteins and associated virus–host interaction and viral pathogenesis. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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Open AccessReview
Emerging Role of LY6E in Virus–Host Interactions
Viruses 2019, 11(11), 1020; https://doi.org/10.3390/v11111020 - 03 Nov 2019
Cited by 10
Abstract
As a canonical lymphocyte antigen-6/urokinase-type plasminogen activator receptor Ly6/uPAR family protein, lymphocyte antigen 6 complex, locus E (LY6E), plays important roles in immunological regulation, T cell physiology, and oncogenesis. Emerging evidence indicates that LY6E is also involved in the modulation of viral infection. [...] Read more.
As a canonical lymphocyte antigen-6/urokinase-type plasminogen activator receptor Ly6/uPAR family protein, lymphocyte antigen 6 complex, locus E (LY6E), plays important roles in immunological regulation, T cell physiology, and oncogenesis. Emerging evidence indicates that LY6E is also involved in the modulation of viral infection. Consequently, viral infection and associated pathogenesis have been associated with altered LY6E gene expression. The interaction between viruses and the host immune system has offered insights into the biology of LY6E. In this review, we summarize the current knowledge of LY6E in the context of viral infection, particularly viral entry. Full article
(This article belongs to the Special Issue Mechanisms of Viral Fusion and Applications in Antivirals)
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