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1
Oliver, Michael R., Kamilla Toon, Charlotte B. Lewis, Stephen Devlin, Robert J. Gifford, and Joe Grove. "Structures of the Hepaci-, Pegi-, and Pestiviruses envelope proteins suggest a novel membrane fusion mechanism." PLOS Biology 21, no. 7 (July 11, 2023): e3002174. http://dx.doi.org/10.1371/journal.pbio.3002174.
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Enveloped viruses encode specialised glycoproteins that mediate fusion of viral and host membranes. Discovery and understanding of the molecular mechanisms of fusion have been achieved through structural analyses of glycoproteins from many different viruses, and yet the fusion mechanisms of some viral genera remain unknown. We have employed systematic genome annotation and AlphaFold modelling to predict the structures of the E1E2 glycoproteins from 60 viral species in the Hepacivirus, Pegivirus, and Pestivirus genera. While the predicted structure of E2 varied widely, E1 exhibited a very consistent fold across genera, despite little or no similarity at the sequence level. Critically, the structure of E1 is unlike any other known viral glycoprotein. This suggests that the Hepaci-, Pegi-, and Pestiviruses may possess a common and novel membrane fusion mechanism. Comparison of E1E2 models from various species reveals recurrent features that are likely to be mechanistically important and sheds light on the evolution of membrane fusion in these viral genera. These findings provide new fundamental understanding of viral membrane fusion and are relevant to structure-guided vaccinology.
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Quinn, Derek J., Neil V. McFerran, John Nelson, and W. Paul Duprex. "Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology." Bioscience Reports 32, no. 3 (March 29, 2012): 333–43. http://dx.doi.org/10.1042/bsr20110100.
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Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein) – haptoEGFPs – which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein–haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.
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Libersou, Sonia, Aurélie A. V. Albertini, Malika Ouldali, Virginie Maury, Christine Maheu, Hélène Raux, Felix de Haas, Stéphane Roche, Yves Gaudin, and Jean Lepault. "Distinct structural rearrangements of the VSV glycoprotein drive membrane fusion." Journal of Cell Biology 191, no. 1 (October 4, 2010): 199–210. http://dx.doi.org/10.1083/jcb.201006116.
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The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes, driven by conformational changes in viral glycoproteins. Many studies have shown that fusion involves the cooperative action of a large number of these glycoproteins, but the underlying mechanisms are unknown. We used electron microscopy and tomography to study the low pH–induced fusion reaction catalyzed by vesicular stomatitis virus glycoprotein (G). Pre- and post-fusion crystal structures were observed on virions at high and low pH, respectively. Individual fusion events with liposomes were also visualized. Fusion appears to be driven by two successive structural rearrangements of G at different sites on the virion. Fusion is initiated at the flat base of the particle. Glycoproteins located outside the contact zone between virions and liposomes then reorganize into regular arrays. We suggest that the formation of these arrays, which have been shown to be an intrinsic property of the G ectodomain, induces membrane constraints, achieving the fusion reaction.
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Lay Mendoza, Maria Fernanda, Marissa Danielle Acciani, Courtney Nina Levit, Christopher Santa Maria, and Melinda Ann Brindley. "Monitoring Viral Entry in Real-Time Using a Luciferase Recombinant Vesicular Stomatitis Virus Producing SARS-CoV-2, EBOV, LASV, CHIKV, and VSV Glycoproteins." Viruses 12, no. 12 (December 17, 2020): 1457. http://dx.doi.org/10.3390/v12121457.
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Viral entry is the first stage in the virus replication cycle and, for enveloped viruses, is mediated by virally encoded glycoproteins. Viral glycoproteins have different receptor affinities and triggering mechanisms. We employed vesicular stomatitis virus (VSV), a BSL-2 enveloped virus that can incorporate non-native glycoproteins, to examine the entry efficiencies of diverse viral glycoproteins. To compare the glycoprotein-mediated entry efficiencies of VSV glycoprotein (G), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S), Ebola (EBOV) glycoprotein (GP), Lassa (LASV) GP, and Chikungunya (CHIKV) envelope (E) protein, we produced recombinant VSV (rVSV) viruses that produce the five glycoproteins. The rVSV virions encoded a nano luciferase (NLucP) reporter gene fused to a destabilization domain (PEST), which we used in combination with the live-cell substrate EndurazineTM to monitor viral entry kinetics in real time. Our data indicate that rVSV particles with glycoproteins that require more post-internalization priming typically demonstrate delayed entry in comparison to VSV G. In addition to determining the time required for each virus to complete entry, we also used our system to evaluate viral cell surface receptor preferences, monitor fusion, and elucidate endocytosis mechanisms. This system can be rapidly employed to examine diverse viral glycoproteins and their entry requirements.
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Jambunathan, Nithya, Carolyn M. Clark, Farhana Musarrat, Vladimir N. Chouljenko, Jared Rudd, and Konstantin G. Kousoulas. "Two Sides to Every Story: Herpes Simplex Type-1 Viral Glycoproteins gB, gD, gH/gL, gK, and Cellular Receptors Function as Key Players in Membrane Fusion." Viruses 13, no. 9 (September 16, 2021): 1849. http://dx.doi.org/10.3390/v13091849.
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Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are prototypical alphaherpesviruses that are characterized by their unique properties to infect trigeminal and dorsal root ganglionic neurons, respectively, and establish life-long latent infections. These viruses initially infect mucosal epithelial tissues and subsequently spread to neurons. They are associated with a significant disease spectrum, including orofacial and ocular infections for HSV-1 and genital and neonatal infections for HSV-2. Viral glycoproteins within the virion envelope bind to specific cellular receptors to mediate virus entry into cells. This is achieved by the fusion of the viral envelope with the plasma membrane. Similarly, viral glycoproteins expressed on cell surfaces mediate cell-to-cell fusion and facilitate virus spread. An interactive complex of viral glycoproteins gB, gD/gH/gL, and gK and other proteins mediate these membrane fusion phenomena with glycoprotein B (gB), the principal membrane fusogen. The requirement for the virion to enter neuronal axons suggests that the heterodimeric protein complex of gK and membrane protein UL20, found only in alphaherpesviruses, constitute a critical determinant for neuronal entry. This hypothesis was substantiated by the observation that a small deletion in the amino terminus of gK prevents entry into neuronal axons while allowing entry into other cells via endocytosis. Cellular receptors and receptor-mediated signaling synergize with the viral membrane fusion machinery to facilitate virus entry and intercellular spread. Unraveling the underlying interactions among viral glycoproteins, envelope proteins, and cellular receptors will provide new innovative approaches for antiviral therapy against herpesviruses and other neurotropic viruses.
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Zhang, You, Joanne York, Melinda A. Brindley, Jack H. Nunberg, and Gregory B. Melikyan. "Fusogenic structural changes in arenavirus glycoproteins are associated with viroporin activity." PLOS Pathogens 19, no. 7 (July 26, 2023): e1011217. http://dx.doi.org/10.1371/journal.ppat.1011217.
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Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.
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Jackson, Julia O., and Richard Longnecker. "Reevaluating Herpes Simplex Virus Hemifusion." Journal of Virology 84, no. 22 (September 15, 2010): 11814–21. http://dx.doi.org/10.1128/jvi.01615-10.
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ABSTRACT Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.
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Melder, Deborah C., Xueqian Yin, Sue E. Delos, and Mark J. Federspiel. "A Charged Second-Site Mutation in the Fusion Peptide Rescues Replication of a Mutant Avian Sarcoma and Leukosis Virus Lacking Critical Cysteine Residues Flanking the Internal Fusion Domain." Journal of Virology 83, no. 17 (June 10, 2009): 8575–86. http://dx.doi.org/10.1128/jvi.00526-09.
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ABSTRACT The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.
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Yang, Xinzhen, Svetla Kurteva, Xinping Ren, Sandra Lee, and Joseph Sodroski. "Subunit Stoichiometry of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers during Virus Entry into Host Cells." Journal of Virology 80, no. 9 (May 1, 2006): 4388–95. http://dx.doi.org/10.1128/jvi.80.9.4388-4395.2006.
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ABSTRACT The envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) function as a homotrimer of gp120/gp41 heterodimers to support virus entry. During the process of virus entry, an individual HIV-1 envelope glycoprotein trimer binds the cellular receptors CD4 and CCR5/CXCR4 and mediates the fusion of the viral and the target cellular membranes. By studying the function of heterotrimers between wild-type and nonfunctional mutant envelope glycoproteins, we found that two wild-type subunits within an envelope glycoprotein trimer are required to support virus entry. Complementation between HIV-1 envelope glycoprotein mutants defective in different functions to allow virus entry was not evident. These results assist our understanding of the mechanisms whereby the HIV-1 envelope glycoproteins mediate virus entry and membrane fusion and guide attempts to inhibit these processes.
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Kinzler, Eric R., and Teresa Compton. "Characterization of Human Cytomegalovirus Glycoprotein-Induced Cell-Cell Fusion." Journal of Virology 79, no. 12 (June 15, 2005): 7827–37. http://dx.doi.org/10.1128/jvi.79.12.7827-7837.2005.
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ABSTRACT Human cytomegalovirus (CMV) infection is dependent on the functions of structural glycoproteins at multiple stages of the viral life cycle. These proteins mediate the initial attachment and fusion events that occur between the viral envelope and a host cell membrane, as well as virion-independent cell-cell spread of the infection. Here we have utilized a cell-based fusion assay to identify the fusogenic glycoproteins of CMV. To deliver the glycoprotein genes to various cell lines, we constructed recombinant retroviruses encoding gB, gH, gL, and gO. Cells expressing individual CMV glycoproteins did not form multinucleated syncytia. Conversely, cells expressing gH/gL showed pronounced syncytium formation, although expression of gH or gL alone had no effect. Anti-gH neutralizing antibodies prevented syncytium formation. Coexpression of gB and/or gO with gH/gL did not yield detectably increased numbers of syncytia. For verification, these results were recapitulated in several cell lines. Additionally, we found that fusion was cell line dependent, as nonimmortalized fibroblast strains did not fuse under any conditions. Thus, the CMV gH/gL complex has inherent fusogenic activity that can be measured in certain cell lines; however, fusion in fibroblast strains may involve a more complex mechanism involving additional viral and/or cellular factors.
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Vallbracht, Melina, Barbara G. Klupp, and Thomas C. Mettenleiter. "Die komplexe Fusionsmaschinerie der Herpesviren." BIOspektrum 28, no. 2 (March 2022): 168–70. http://dx.doi.org/10.1007/s12268-022-1718-5.
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AbstractEnveloped viruses enter cells by fusion between viral and cellular membranes which is catalyzed by specialized fusogenic glycoproteins (g) on the viral surface. Many viruses use a single fusion protein for entry. In contrast, herpesviruses depend on a complex fusion machinery. Here, we discuss the role of the individual herpesvirus fusion machinery components and answer two basic questions: why does the herpesvirus fusion protein gB depend on other glycoproteins for fusion, and can gB be transformed to function autonomously?
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Lavillette, Dimitri, Alessia Ruggieri, Bertrand Boson, Marielle Maurice, and François-Loïc Cosset. "Relationship between SU Subdomains That Regulate the Receptor-Mediated Transition from the Native (Fusion-Inhibited) to the Fusion-Active Conformation of the Murine Leukemia Virus Glycoprotein." Journal of Virology 76, no. 19 (October 1, 2002): 9673–85. http://dx.doi.org/10.1128/jvi.76.19.9673-9685.2002.
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ABSTRACT Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All these different mutations call for a critical role of the PRR in mediating conformational changes of the Env glycoprotein during fusion activation. Our results suggest a model of MLV Env fusion activation in which unlocking of the fusion-inhibitory conformation is initiated by receptor binding of the viral RBD, which, upon disruption of the PRR, allows the NTR domain to promote further events in Env fusion activation. This involves a second type of interaction, in cis or in trans, between the receptor-activated RBD and a median segment of the freed C-terminal domain.
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Bude, Sara Amanuel, Zengjun Lu, Zhixun Zhao, and Qiang Zhang. "Pseudorabies Virus Glycoproteins E and B Application in Vaccine and Diagnosis Kit Development." Vaccines 12, no. 9 (September 20, 2024): 1078. http://dx.doi.org/10.3390/vaccines12091078.
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Background: Pseudorabies virus (PRV) is a highly infectious pathogen that affects a wide range of mammals and imposes a significant economic burden on the global pig industry. The viral envelope of PRV contains several glycoproteins, including glycoprotein E (gE) and glycoprotein B (gB), which play critical roles in immune recognition, vaccine development, and diagnostic procedures. Mutations in these glycoproteins may enhance virulence, highlighting the need for updated vaccines. Method: This review examines the functions of PRV gE and gB in vaccine development and diagnostics, focusing on their roles in viral replication, immune system interaction, and pathogenicity. Additionally, we explore recent findings on the importance of gE deletion in attenuated vaccines and the potential of gB to induce immunity. Results: Glycoprotein E (gE) is crucial for the virus’s axonal transport and nerve invasion, facilitating transmission to the central nervous system. Deletion of gE is a successful strategy in vaccine development, enhancing the immune response. Glycoprotein B (gB) plays a central role in viral replication and membrane fusion, aiding viral spread. Mutations in these glycoproteins may increase PRV virulence, complicating vaccine efficacy. Conclusion: With PRV glycoproteins being essential to both vaccine development and diagnostic approaches, future research should focus on enhancing these components to address emerging PRV variants. Updated vaccines and diagnostic tools are critical for combating new, more virulent strains of PRV.
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Garry, Courtney E., and Robert F. Garry. "Proteomics Computational Analyses Suggest that the Antennavirus Glycoprotein Complex Includes a Class I Viral Fusion Protein (α-Penetrene) with an Internal Zinc-Binding Domain and a Stable Signal Peptide." Viruses 11, no. 8 (August 14, 2019): 750. http://dx.doi.org/10.3390/v11080750.
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A metatranscriptomic study of RNA viruses in cold-blooded vertebrates identified two related viruses from frogfish (Antennarius striatus) that represent a new genus Antennavirus in the family Arenaviridae (Order: Bunyavirales). Computational analyses were used to identify features common to class I viral fusion proteins (VFPs) in antennavirus glycoproteins, including an N-terminal fusion peptide, two extended alpha-helices, an intrahelical loop, and a carboxyl terminal transmembrane domain. Like mammarenavirus and hartmanivirus glycoproteins, the antennavirus glycoproteins have an intracellular zinc-binding domain and a long virion-associated stable signal peptide (SSP). The glycoproteins of reptarenaviruses are also class I VFPs, but do not contain zinc-binding domains nor do they encode SSPs. Divergent evolution from a common progenitor potentially explains similarities of antennavirus, mammarenavirus, and hartmanivirus glycoproteins, with an ancient recombination event resulting in a divergent reptarenavirus glycoprotein.
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Earnest, James T., Michael P. Hantak, Jung-Eun Park, and Tom Gallagher. "Coronavirus and Influenza Virus Proteolytic Priming Takes Place in Tetraspanin-Enriched Membrane Microdomains." Journal of Virology 89, no. 11 (April 1, 2015): 6093–104. http://dx.doi.org/10.1128/jvi.00543-15.
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ABSTRACTCoronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases.IMPORTANCEEnveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus priming with greater precision. Implications of these findings extend to the use of virus entry antagonists, such as protease inhibitors, which might be most effective when localized to these microdomains.
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Fan, Qing, Richard Longnecker, and Sarah A. Connolly. "A Functional Interaction between Herpes Simplex Virus 1 Glycoprotein gH/gL Domains I and II and gD Is Defined by Using Alphaherpesvirus gH and gL Chimeras." Journal of Virology 89, no. 14 (April 29, 2015): 7159–69. http://dx.doi.org/10.1128/jvi.00740-15.
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ABSTRACTWhereas most viruses require only a single protein to bind to and fuse with cells, herpesviruses use multiple glycoproteins to mediate virus entry, and thus communication among these proteins is required. For most alphaherpesviruses, the minimal set of viral proteins required for fusion with the host cell includes glycoproteins gD, gB, and a gH/gL heterodimer. In the current model of entry, gD binds to a cellular receptor and transmits a signal to gH/gL. This signal then triggers gB, the conserved fusion protein, to insert into the target membrane and refold to merge the viral and cellular membranes. We previously demonstrated that gB homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1), were interchangeable. In contrast, neither gD nor gH/gL functioned with heterotypic entry glycoproteins, indicating that gD and gH/gL exhibit an essential type-specific functional interaction. To map this homotypic interaction site on gH/gL, we generated HSV-1/SaHV-1 gH and gL chimeras. The functional interaction with HSV-1 gD mapped to the N-terminal domains I and II of the HSV-1 gH ectodomain. The core of HSV-1 gL that interacts with gH also was required for functional homotypic interaction. The N-terminal gH/gL domains I and II are the least conserved and may have evolved to support species-specific glycoprotein interactions.IMPORTANCEThe first step of the herpesvirus life cycle is entry into a host cell. A coordinated interaction among multiple viral glycoproteins is required to mediate fusion of the viral envelope with the cell membrane. The details of how these glycoproteins interact to trigger fusion are unclear. By swapping the entry glycoproteins of two alphaherpesviruses (HSV-1 and SaHV-1), we previously demonstrated a functional homotypic interaction between gD and gH/gL. To define the gH and gL requirements for homotypic interaction, we evaluated the function of a panel of HSV-1/SaHV-1 gH and gL chimeras. We demonstrate that domains I and II of HSV-1 gH are sufficient to promote a functional, albeit reduced, interaction with HSV-1 gD. These findings contribute to our model of how the entry glycoproteins cooperate to mediate herpesvirus entry into the cell.
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Federspiel, Mark J. "Reverse Engineering Provides Insights on the Evolution of Subgroups A to E Avian Sarcoma and Leukosis Virus Receptor Specificity." Viruses 11, no. 6 (May 30, 2019): 497. http://dx.doi.org/10.3390/v11060497.
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The initial step of retrovirus entry—the interaction between the virus envelope glycoprotein trimer and a cellular receptor—is complex, involving multiple, noncontiguous determinants in both proteins that specify receptor choice, binding affinity and the ability to trigger conformational changes in the viral glycoproteins. Despite the complexity of this interaction, retroviruses have the ability to evolve the structure of their envelope glycoproteins to use a different cellular protein as receptors. The highly homologous subgroup A to E Avian Sarcoma and Leukosis Virus (ASLV) glycoproteins belong to the group of class 1 viral fusion proteins with a two-step triggering mechanism that allows experimental access to intermediate structures during the fusion process. We and others have taken advantage of replication-competent ASLVs and exploited genetic selection strategies to force the ASLVs to naturally evolve and acquire envelope glycoprotein mutations to escape the pressure on virus entry and still yield a functional replicating virus. This approach allows for the simultaneous selection of multiple mutations in multiple functional domains of the envelope glycoprotein that may be required to yield a functional virus. Here, we review the ASLV family and experimental system and the reverse engineering approaches used to understand the evolution of ASLV receptor usage.
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Snyder, Aleksandra, Todd W. Wisner, and David C. Johnson. "Herpes Simplex Virus Capsids Are Transported in Neuronal Axons without an Envelope Containing the Viral Glycoproteins." Journal of Virology 80, no. 22 (September 13, 2006): 11165–77. http://dx.doi.org/10.1128/jvi.01107-06.
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ABSTRACT Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini.
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Bowden, Thomas A., Max Crispin, Stephen C. Graham, David J. Harvey, Jonathan M. Grimes, E. Yvonne Jones, and David I. Stuart. "Unusual Molecular Architecture of the Machupo Virus Attachment Glycoprotein." Journal of Virology 83, no. 16 (June 3, 2009): 8259–65. http://dx.doi.org/10.1128/jvi.00761-09.
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ABSTRACT New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel α/β fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.
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Tischler, Nicole D., Angel Gonzalez, Tomas Perez-Acle, Mario Rosemblatt, and Pablo D. T. Valenzuela. "Hantavirus Gc glycoprotein: evidence for a class II fusion protein." Journal of General Virology 86, no. 11 (November 1, 2005): 2937–47. http://dx.doi.org/10.1099/vir.0.81083-0.
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Hantavirus cell entry is promoted by its envelope glycoproteins, Gn and Gc, through cell attachment and by fusion between viral and endosomal membranes at low pH. However, the role of Gn and Gc in receptor binding and cell fusion has not yet been defined. In this work, a sequence presenting characteristics similar to those of class II fusion peptides (FPs) of alphavirus E1 and flavivirus E proteins is identified within the hantavirus Gc glycoprotein. A three-dimensional comparative molecular model based on crystallographic data of tick-borne encephalitis virus E protein is proposed for the Andes virus (ANDV) Gc ectodomain, which supports a feasible class II fusion-protein fold. In vitro experimental evidence is provided for the binding activity of the ANDV FP candidate to artificial membranes, as demonstrated by fluorescence anisotropy assays. Taken together, these results support the hypothesis that the Gc glycoprotein of hantaviruses and of other members of the family Bunyaviridae directs the viral fusion activity and that it may be classified as a class II viral fusion protein.
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Snyder, Aleksandra, Birgitte Bruun, Helena M. Browne, and David C. Johnson. "A Herpes Simplex Virus gD-YFP Fusion Glycoprotein Is Transported Separately from Viral Capsids in Neuronal Axons." Journal of Virology 81, no. 15 (May 23, 2007): 8337–40. http://dx.doi.org/10.1128/jvi.00520-07.
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ABSTRACT Two models describing how alphaherpesviruses exit neurons differ with respect to whether nucleocapsids and envelope glycoproteins travel toward axon termini separately or as assembled enveloped virions. Recently, a pseudorabies virus glycoprotein D (gD)-green fluorescent protein fusion was found to colocalize with viral capsids, supporting anterograde transport of enveloped virions. Previous antibody staining experiments demonstrated that herpes simplex virus (HSV) glycoproteins and capsids are separately transported in axons. Here, we generated an HSV expressing a gD-yellow fluorescent protein (YFP) fusion and found that gD-YFP and capsids were transported separately in neuronal axons. Anti-gD antibodies colocalized with gD-YFP, indicating that gD-YFP behaves like wild-type HSV gD.
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Drummer, Heidi E., Irene Boo, and Pantelis Poumbourios. "Mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis C virus glycoprotein E1." Journal of General Virology 88, no. 4 (April 1, 2007): 1144–48. http://dx.doi.org/10.1099/vir.0.82567-0.
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The E1E2 glycoprotein heterodimer of Hepatitis C virus mediates viral entry. E2 attaches the virus to cellular receptors; however, the function of E1 is unknown. We tested the hypothesis that E1 is a truncated class II fusion protein. We mutated amino acids within a predicted fusion peptide (residues 276–286) and a truncated C-terminal stem-like motif, containing a membrane-proximal heptad-repeat sequence (residues 330–347). The fusion peptide mutation F285A abolished viral entry, while mutation of other hydrophobic residues had no effect. Alanine replacement of heptad-repeat residues blocked entry in three of five cases, whereas substitution with the helix breaker, Pro, led to loss of entry function in all cases. The mutations did not affect glycoprotein expression, heterodimerization with E2 or global folding, in contrast to the effects of mutations in the fusion motifs of prototypical class II fusion proteins. Our data suggest that E1 is unlikely to function in an analogous manner to other class II fusion glycoproteins.
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Santos, Joy Ramielle L., Weijie Sun, Tarana A. Mangukia, Eduardo Reyes-Serratos, and Marcelo Marcet-Palacios. "Challenging the Existing Model of the Hexameric HIV-1 Gag Lattice and MA Shell Superstructure: Implications for Viral Entry." Viruses 13, no. 8 (July 31, 2021): 1515. http://dx.doi.org/10.3390/v13081515.
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Despite type 1 human immunodeficiency virus (HIV-1) being discovered in the early 1980s, significant knowledge gaps remain in our understanding of the superstructure of the HIV-1 matrix (MA) shell. Current viral assembly models assume that the MA shell originates via recruitment of group-specific antigen (Gag) polyproteins into a hexagonal lattice but fails to resolve and explain lattice overlapping that occurs when the membrane is folded into a spherical/ellipsoidal shape. It further fails to address how the shell recruits, interacts with and encompasses the viral spike envelope (Env) glycoproteins. These Env glycoproteins are crucial as they facilitate viral entry by interacting with receptors and coreceptors located on T-cells. In our previous publication, we proposed a six-lune hosohedral structure, snowflake-like model for the MA shell of HIV-1. In this article, we improve upon the six-lune hosohedral structure by incorporating into our algorithm the recruitment of complete Env glycoproteins. We generated the Env glycoprotein assembly using a combination of predetermined Env glycoprotein domains from X-ray crystallography, nuclear magnetic resonance (NMR), cryoelectron tomography, and three-dimensional prediction tools. Our novel MA shell model comprises 1028 MA trimers and 14 Env glycoproteins. Our model demonstrates the movement of Env glycoproteins in the interlunar spaces, with effective clustering at the fusion hub, where multiple Env complexes bind to T-cell receptors during the process of viral entry. Elucidating the HIV-1 MA shell structure and its interaction with the Env glycoproteins is a key step toward understanding the mechanism of HIV-1 entry.
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Stone, Jacquelyn A., Bhadra M. Vemulapati, Birgit Bradel-Tretheway, and Hector C. Aguilar. "Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins." Journal of Virology 90, no. 23 (September 21, 2016): 10762–73. http://dx.doi.org/10.1128/jvi.01469-16.
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ABSTRACTThe paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions.IMPORTANCENipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family.
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Bradel-Tretheway, Birgit G., Qian Liu, Jacquelyn A. Stone, Samantha McInally, and Hector C. Aguilar. "Novel Functions of Hendra Virus G N-Glycans and Comparisons to Nipah Virus." Journal of Virology 89, no. 14 (May 6, 2015): 7235–47. http://dx.doi.org/10.1128/jvi.00773-15.
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ABSTRACTHendra virus (HeV) and Nipah virus (NiV) are reportedly the most deadly pathogens within theParamyxoviridaefamily. These two viruses bind the cellular entry receptors ephrin B2 and/or ephrin B3 via the viral attachment glycoprotein G, and the concerted efforts of G and the viral fusion glycoprotein F result in membrane fusion. Membrane fusion is essential for viral entry into host cells and for cell-cell fusion, a hallmark of the disease pathobiology. HeV G is heavily N-glycosylated, but the functions of the N-glycans remain unknown. We disrupted eight predicted N-glycosylation sites in HeV G by conservative mutations (Asn to Gln) and found that six out of eight sites were actually glycosylated (G2 to G7); one in the stalk (G2) and five in the globular head domain (G3 to G7). We then tested the roles of individual and combined HeV G N-glycan mutants and found functions in the modulation of shielding against neutralizing antibodies, intracellular transport, G-F interactions, cell-cell fusion, and viral entry. Between the highly conserved HeV and NiV G glycoproteins, similar trends in the effects of N-glycans on protein functions were observed, with differences in the levels at which some N-glycan mutants affected such functions. While the N-glycan in the stalk domain (G2) had roles that were highly conserved between HeV and NiV G, individual N-glycans in the head affected the levels of several protein functions differently. Our findings are discussed in the context of their contributions to our understanding of HeV and NiV pathogenesis and immune responses.IMPORTANCEViral envelope glycoproteins are important for viral pathogenicity and immune evasion. N-glycan shielding is one mechanism by which immune evasion can be achieved. In paramyxoviruses, viral attachment and membrane fusion are governed by the close interaction of the attachment proteins H/HN/G and the fusion protein F. In this study, we show that the attachment glycoprotein G of Hendra virus (HeV), a deadly paramyxovirus, is N-glycosylated at six sites (G2 to G7) and that most of these sites have important roles in viral entry, cell-cell fusion, G-F interactions, G oligomerization, and immune evasion. Overall, we found that the N-glycan in the stalk domain (G2) had roles that were very conserved between HeV G and the closely related Nipah virus G, whereas individual N-glycans in the head quantitatively modulated several protein functions differently between the two viruses.
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Dollery, Stephen J. "Towards Understanding KSHV Fusion and Entry." Viruses 11, no. 11 (November 18, 2019): 1073. http://dx.doi.org/10.3390/v11111073.
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How viruses enter cells is of critical importance to pathogenesis in the host and for treatment strategies. Over the last several years, the herpesvirus field has made numerous and thoroughly fascinating discoveries about the entry of alpha-, beta-, and gamma-herpesviruses, giving rise to knowledge of entry at the amino acid level and the realization that, in some cases, researchers had overlooked whole sets of molecules essential for entry into critical cell types. Herpesviruses come equipped with multiple envelope glycoproteins which have several roles in many aspects of infection. For herpesvirus entry, it is usual that a collective of glycoproteins is involved in attachment to the cell surface, specific interactions then take place between viral glycoproteins and host cell receptors, and then molecular interactions and triggers occur, ultimately leading to viral envelope fusion with the host cell membrane. The fact that there are multiple cell and virus molecules involved with the build-up to fusion enhances the diversity and specificity of target cell types, the cellular entry pathways the virus commandeers, and the final triggers of fusion. This review will examine discoveries relating to how Kaposi’s sarcoma-associated herpesvirus (KSHV) encounters and binds to critical cell types, how cells internalize the virus, and how the fusion may occur between the viral membrane and the host cell membrane. Particular focus is given to viral glycoproteins and what is known about their mechanisms of action.
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Hasegawa, Kosei, Chunling Hu, Takafumi Nakamura, James D. Marks, Stephen J. Russell, and Kah-Whye Peng. "Affinity Thresholds for Membrane Fusion Triggering by Viral Glycoproteins." Journal of Virology 81, no. 23 (September 5, 2007): 13149–57. http://dx.doi.org/10.1128/jvi.01415-07.
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ABSTRACT Enveloped viruses trigger membrane fusion to gain entry into cells. The receptor affinities of their attachment proteins vary greatly, from 10−4 M to 10−9 M, but the significance of this is unknown. Using six retargeted measles viruses that bind to Her-2/neu with a 5-log range in affinity, we show that receptor affinity has little impact on viral attachment but is nevertheless a key determinant of infectivity and intercellular fusion. For a given cell surface receptor density, there is an affinity threshold above which cell-cell fusion proceeds efficiently. Suprathreshold affinities do not further enhance the efficiency of membrane fusion.
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Batonick, Melissa, Antonius G. P. Oomens, and Gail W. Wertz. "Human Respiratory Syncytial Virus Glycoproteins Are Not Required for Apical Targeting and Release from Polarized Epithelial Cells." Journal of Virology 82, no. 17 (June 18, 2008): 8664–72. http://dx.doi.org/10.1128/jvi.00827-08.
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ABSTRACT Human respiratory syncytial virus (HRSV) is released from the apical membrane of polarized epithelial cells. However, little is known about the processes of assembly and release of HRSV and which viral gene products are involved in the directional maturation of the virus. Based on previous studies showing that the fusion (F) glycoprotein contained an intrinsic apical sorting signal and that N- and O-linked glycans can act as apical targeting signals, we investigated whether the glycoproteins of HRSV were involved in its directional targeting and release. We generated recombinant viruses with each of the three glycoprotein genes deleted individually or in groups. Each deleted gene was replaced with a reporter gene to maintain wild-type levels of gene expression. The effects of deleting the glycoprotein genes on apical maturation and on targeting of individual proteins in polarized epithelial cells were examined by using biological, biochemical, and microscopic assays. The results of these studies showed that the HRSV glycoproteins are not required for apical maturation or release of the virus. Further, deletion of one or more of the glycoprotein genes did not affect the intracellular targeting of the remaining viral glycoproteins or the nucleocapsid protein to the apical membrane.
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Zhao, Xuesen, Fang Guo, Mary Ann Comunale, Anand Mehta, Mohit Sehgal, Pooja Jain, Andrea Cuconati, et al. "Inhibition of Endoplasmic Reticulum-Resident Glucosidases Impairs Severe Acute Respiratory Syndrome Coronavirus and Human Coronavirus NL63 Spike Protein-Mediated Entry by Altering the Glycan Processing of Angiotensin I-Converting Enzyme 2." Antimicrobial Agents and Chemotherapy 59, no. 1 (October 27, 2014): 206–16. http://dx.doi.org/10.1128/aac.03999-14.
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ABSTRACTEndoplasmic reticulum (ER)-resident glucosidases I and II sequentially trim the three terminal glucose moieties on the N-linked glycans attached to nascent glycoproteins. These reactions are the first steps of N-linked glycan processing and are essential for proper folding and function of many glycoproteins. Because most of the viral envelope glycoproteins contain N-linked glycans, inhibition of ER glucosidases with derivatives of 1-deoxynojirimycin, i.e., iminosugars, efficiently disrupts the morphogenesis of a broad spectrum of enveloped viruses. However, like viral envelope proteins, the cellular receptors of many viruses are also glycoproteins. It is therefore possible that inhibition of ER glucosidases not only compromises virion production but also disrupts expression and function of viral receptors and thus inhibits virus entry into host cells. Indeed, we demonstrate here that iminosugar treatment altered the N-linked glycan structure of angiotensin I-converting enzyme 2 (ACE2), which did not affect its expression on the cell surface or its binding of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein. However, alteration of N-linked glycans of ACE2 impaired its ability to support the transduction of SARS-CoV and human coronavirus NL63 (HCoV-NL63) spike glycoprotein-pseudotyped lentiviral particles by disruption of the viral envelope protein-triggered membrane fusion. Hence, in addition to reducing the production of infectious virions, inhibition of ER glucosidases also impairs the entry of selected viruses via a post-receptor-binding mechanism.
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Piñón, Josefina D., Sharon M. Kelly, Nicholas C. Price, Jack U. Flanagan, and David W. Brighty. "An Antiviral Peptide Targets a Coiled-Coil Domain of the Human T-Cell Leukemia Virus Envelope Glycoprotein." Journal of Virology 77, no. 5 (March 1, 2003): 3281–90. http://dx.doi.org/10.1128/jvi.77.5.3281-3290.2003.
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ABSTRACT Retrovirus entry into cells is mediated by the viral envelope glycoproteins which, through a cascade of conformational changes, orchestrate fusion of the viral and cellular membranes. In the absence of membrane fusion, viral entry into the host cell cannot occur. For human T-cell leukemia virus type 1 (HTLV-1), synthetic peptides that mimic a carboxy-terminal region of the transmembrane glycoprotein (TM) ectodomain are potent inhibitors of membrane fusion and virus entry. Here, we demonstrate that this class of inhibitor targets a fusion-active structure of HTLV-1 envelope. In particular, the peptides bind specifically to a core coiled-coil domain of envelope, and peptide variants that fail to bind the coiled-coil lack inhibitory activity. Our data indicate that the inhibitory peptides likely function by disrupting the formation of a trimer-of-hairpins structure that is required for membrane fusion. Importantly, we also show that peptides exhibiting dramatically increased potency can be readily obtained. We suggest that peptides or peptide mimetics targeting the fusion-active structures of envelope may be of therapeutic value in the treatment of HTLV-1 infections.
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Nieva, José L., and Tatiana Suárez. "Hydrophobic-at-Interface Regions in Viral Fusion Protein Ectodomains." Bioscience Reports 20, no. 6 (December 1, 2000): 519–33. http://dx.doi.org/10.1023/a:1010458904487.
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In this chapter we shall describe how to apply the hydrophobicity-at-interface scale, as proposed by Wimley and White [Wimley, W. C. and White, S. H. (1996) Nature Struct. Biol. 3:842–848], to the detection of amino acid sequences of viral envelope glycoproteins putatively engaged in interactions with the target membranes. In addition, a new approach will be briefly introduced to infer the bilayer location at equilibrium of membrane-partitioning sequences. The use of these new procedures may be important in describing the molecular mechanism leading to the formation of a fusion pore by viral glycoproteins.
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Chandra, Sunandini, Raju Kalaivani, Manoj Kumar, Narayanaswamy Srinivasan, and Debi P. Sarkar. "Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes." Molecular Biology of the Cell 28, no. 26 (December 15, 2017): 3801–14. http://dx.doi.org/10.1091/mbc.e17-06-0400.
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Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion–mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206—an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.
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Garry, Courtney, and Robert Garry. "Proteomics Computational Analyses Suggest that the Envelope Glycoproteins of Segmented Jingmen Flavi-Like Viruses Are Class II Viral Fusion Proteins (β-Penetrenes) with Mucin-Like Domains." Viruses 12, no. 3 (February 27, 2020): 260. http://dx.doi.org/10.3390/v12030260.
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Jingmen viruses are newly described segmented flavi-like viruses that have a worldwide distribution in ticks and have been associated with febrile illnesses in humans. Computational analyses were used to predict that Jingmen flavi-like virus glycoproteins have structural features of class II viral fusion proteins, including an ectodomain consisting of beta-sheets and short alpha-helices, a fusion peptide with interfacial hydrophobicity and a three-domain architecture. Jingmen flavi-like virus glycoproteins have a sequence enriched in serine, threonine, and proline at the amino terminus, which is a feature of mucin-like domains. Several of the serines and threonines are predicted be modified by the addition of O-linked glycans. Some of the glycoproteins are predicted to have an additional mucin-like domain located prior to the transmembrane anchor, whereas others are predicted to have a stem consisting of two alpha-helices. The flavivirus envelope protein and Jingmen flavi-virus glycoproteins may have diverged from a common class II precursor glycoprotein with a mucin-like domain or domains acquired after divergence.
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Chen, Bing, Yifan Cheng, Lesley Calder, Stephen C. Harrison, Ellis L. Reinherz, John J. Skehel, and Don C. Wiley. "A Chimeric Protein of Simian Immunodeficiency Virus Envelope Glycoprotein gp140 and Escherichia coli Aspartate Transcarbamoylase." Journal of Virology 78, no. 9 (May 1, 2004): 4508–16. http://dx.doi.org/10.1128/jvi.78.9.4508-4516.2004.
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ABSTRACT The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R2) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C6R6 ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.
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Balliet, John W., Kristin Gendron, and Paul Bates. "Mutational Analysis of the Subgroup A Avian Sarcoma and Leukosis Virus Putative Fusion Peptide Domain." Journal of Virology 74, no. 8 (April 15, 2000): 3731–39. http://dx.doi.org/10.1128/jvi.74.8.3731-3739.2000.
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ABSTRACT Short hydrophobic regions referred to as fusion peptide domains (FPDs) at or near the amino terminus of the membrane-anchoring subunit of viral glycoproteins are believed to insert into the host membrane during the initial stage of enveloped viral entry. Avian sarcoma and leukosis viruses (ASLV) are unusual among retroviruses in that the region in the envelope glycoprotein (EnvA) proposed to be the FPD is internal and contains a centrally located proline residue. To begin analyzing the function of this region of EnvA, 20 substitution mutations were introduced into the putative FPD. The mutant envelope glycoproteins were evaluated for effects on virion incorporation, receptor binding, and infection. Interestingly, most of the single-substitution mutations had little effect on any of these processes. In contrast, a bulky hydrophobic substitution for the central proline reduced viral titers 15-fold without affecting virion incorporation or receptor binding, whereas substitution of glycine for the proline had only a nominal effect on EnvA function. Similar to other viral FPDs, the putative ASLV FPD has been modeled as an amphipathic helix where most of the bulky hydrophobic residues form a patch on one face of the helix. A series of alanine insertion mutations designed to interrupt the hydrophobic patch on the helix had differential effects on infectivity, and the results of that analysis together with the results observed with the substitution mutations suggest no correlation between maintenance of the hydrophobic patch and glycoprotein function.
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Chi, Jung Hee I., Carol A. Harley, Aparna Mukhopadhyay, and Duncan W. Wilson. "The cytoplasmic tail of herpes simplex virus envelope glycoprotein D binds to the tegument protein VP22 and to capsids." Journal of General Virology 86, no. 2 (February 1, 2005): 253–61. http://dx.doi.org/10.1099/vir.0.80444-0.
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Herpes simplex virus (HSV) capsids assemble, mature and package their viral genome in the nucleoplasm. They then exit the nucleus into the cytoplasm, where they acquire their final tegument and envelope. The molecular mechanism of cytoplasmic envelopment is unclear, but evidence suggests that the viral glycoprotein tails play an important role in the recruitment of tegument and capsids at the final envelopment site. However, due to redundancy in protein–protein interactions among the viral glycoproteins, genetic analysis of the role of individual glycoproteins in assembly has been difficult. To overcome this problem, a glutathione S-transferase fusion protein-binding assay was used in this study to test the interaction between the cytoplasmic tail of one specific viral glycoprotein, gD, and tegument proteins. The study demonstrated that the 38 kDa tegument protein VP22 bound specifically to the gD tail. This association was dependent on arginine and lysine residues at positions 5 and 6 in the gD tail. In addition, HSV-1 capsids bound the gD tail and exhibited a similar sequence dependence. It is concluded that VP22 may serve as a linker protein, mediating the interaction of the HSV capsid with gD.
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Klupp, Barbara G., Ralf Nixdorf, and Thomas C. Mettenleiter. "Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion." Journal of Virology 74, no. 15 (August 1, 2000): 6760–68. http://dx.doi.org/10.1128/jvi.74.15.6760-6768.2000.
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ABSTRACT A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873–875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014–3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.
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De Clercq, Erik. "Antiviral Metal Complexes." Metal-Based Drugs 4, no. 3 (January 1, 1997): 173–92. http://dx.doi.org/10.1155/mbd.1997.173.
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The initial events (virus adsorption and fusion with the cells) in the replicative cycle of human immunodeficiency virus (HIV) can serve as targets for the antiviral action of metal-binding compounds such as polyanionic compounds (polysulfates, polysulfonates, polycarboxylates, polyoxometalates, and sulfonated or carboxylated metalloporphyrins), bicyclams and G-octet-forming oligonucleotides. The adsorption and fusion of HIV with its target cells depends on the interaction of the viral envelope glycoproteins (gp 120) with the receptors (CD4, CXCR4) at the outer cell membrane. We are currently investigating how the aforementioned compounds interfere with these viral glycoproteins and/or cell receptor.
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Plemper, Richard K., Anthea L. Hammond, Denis Gerlier, Adele K. Fielding, and Roberto Cattaneo. "Strength of Envelope Protein Interaction Modulates Cytopathicity of Measles Virus." Journal of Virology 76, no. 10 (May 15, 2002): 5051–61. http://dx.doi.org/10.1128/jvi.76.10.5051-5061.2002.
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ABSTRACT To understand the molecular determinants of measles virus (MV) cytopathicity, we have characterized mutant viruses exhibiting a more-extensive cell-to-cell fusion while maintaining efficient replication to high titers. A virus which is modified by the addition of an 8-amino-acid Flag epitope tag at the cytoplasmic tail of its H (for MV hemagglutinin) envelope glycoprotein replicates efficiently, has an increased cytopathicity, possesses a greater infectivity per particle, and has an altered protein composition compared with that of unmodified MV. The mutant phenotype is not specifically linked to the epitope sequence, since an alternatively added HA (for influenza virus-derived hemagglutinin) epitope tag caused similar effects. We demonstrate that both epitope tags weaken the interaction between the H and fusion (F) glycoproteins in virus-infected cells. This reduction in strength of H/F interaction is independent of the presence of the viral matrix (M) protein. Viruses with this less stable complex are more sensitive to neutralization by a soluble octameric form of the CD46 receptor, consistent with their increased fusogenicity. Similar analyses of glycoproteins derived from MV strains with reduced cytopathicities confirm that the strength of H and F glycoprotein interaction is a modulator of viral fusogenicity.
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Pertel, Peter E. "Human Herpesvirus 8 Glycoprotein B (gB), gH, and gL Can Mediate Cell Fusion." Journal of Virology 76, no. 9 (May 1, 2002): 4390–400. http://dx.doi.org/10.1128/jvi.76.9.4390-4400.2002.
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ABSTRACT Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gBMUT) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gBMUT was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gBMUT for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins.
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Webb, Stacy R., Stacy E. Smith, Michael G. Fried, and Rebecca Ellis Dutch. "Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro." mSphere 3, no. 2 (April 18, 2018): e00047-18. http://dx.doi.org/10.1128/msphere.00047-18.
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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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Lavillette, Dimitri, Eve-Isabelle Pécheur, Peggy Donot, Judith Fresquet, Jennifer Molle, Romuald Corbau, Marlène Dreux, François Penin, and François-Loïc Cosset. "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 81, no. 16 (May 30, 2007): 8752–65. http://dx.doi.org/10.1128/jvi.02642-06.
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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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Madani, Navid, Amy M. Hubicki, Ana Luisa Perdigoto, Martin Springer, and Joseph Sodroski. "Inhibition of Human Immunodeficiency Virus Envelope Glycoprotein- Mediated Single Cell Lysis by Low-Molecular-Weight Antagonists of Viral Entry." Journal of Virology 81, no. 2 (August 30, 2006): 532–38. http://dx.doi.org/10.1128/jvi.01079-06.
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ABSTRACT The coexpression of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins and receptors leads to the lysis of single cells by a process that is dependent upon membrane fusion. This cell lysis was inhibited by low-molecular-weight compounds that interfere with receptor binding or with receptor-induced conformational transitions in the envelope glycoproteins. A peptide, T20, potently inhibited cell-cell fusion but had no effect on single cell lysis mediated by the HIV-1 envelope glycoproteins. Thus, critical events in the lysis of single cells by the HIV-1 envelope glycoproteins occur in intracellular compartments accessible only to small inhibitory compounds.
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Kelly, James T., Stacey Human, Joseph Alderman, Fatoumatta Jobe, Leanne Logan, Thomas Rix, Daniel Gonçalves-Carneiro, et al. "BST2/Tetherin Overexpression Modulates Morbillivirus Glycoprotein Production to Inhibit Cell–Cell Fusion." Viruses 11, no. 8 (July 30, 2019): 692. http://dx.doi.org/10.3390/v11080692.
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The measles virus (MeV), a member of the genus Morbillivirus, is an established pathogen of humans. A key feature of morbilliviruses is their ability to spread by virus–cell and cell–cell fusion. The latter process, which leads to syncytia formation in vitro and in vivo, is driven by the viral fusion (F) and haemagglutinin (H) glycoproteins. In this study, we demonstrate that MeV glycoproteins are sensitive to inhibition by bone marrow stromal antigen 2 (BST2/Tetherin/CD317) proteins. BST2 overexpression causes a large reduction in MeV syncytia expansion. Using quantitative cell–cell fusion assays, immunolabeling, and biochemistry we further demonstrate that ectopically expressed BST2 directly inhibits MeV cell–cell fusion. This restriction is mediated by the targeting of the MeV H glycoprotein, but not other MeV proteins. Using truncation mutants, we further establish that the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 is required for the restriction of MeV replication in vitro and cell–cell fusion. By extending our study to the ruminant morbillivirus peste des petits ruminants virus (PPRV) and its natural host, sheep, we also confirm this is a broad and cross-species specific phenotype.
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Zhou, Xuan, Giorgia Cimato, Yihua Zhou, Giada Frascaroli, and Wolfram Brune. "A Virus Genetic System to Analyze the Fusogenicity of Human Cytomegalovirus Glycoprotein B Variants." Viruses 15, no. 4 (April 16, 2023): 979. http://dx.doi.org/10.3390/v15040979.
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Viruses can induce the fusion of infected and neighboring cells, leading to the formation of syncytia. Cell–cell fusion is mediated by viral fusion proteins on the plasma membrane of infected cells that interact with cellular receptors on neighboring cells. Viruses use this mechanism to spread rapidly to adjacent cells or escape host immunity. For some viruses, syncytium formation is a hallmark of infection and a known pathogenicity factor. For others, the role of syncytium formation in viral dissemination and pathogenicity remains poorly understood. Human cytomegalovirus (HCMV) is an important cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. Clinical HCMV isolates have broad cell tropism but differ in their ability to induce cell–cell fusions, and little is known about the molecular determinants. We developed a system to analyze HCMV glycoprotein B (gB) variants in a defined genetic background. HCMV strains TB40/E and TR were used as vectors to compare the fusogenicity of six gB variants from congenitally infected fetuses with those from three laboratory strains. Five of them conferred the ability to induce the fusion of MRC-5 human embryonic lung fibroblasts to one or both backbone strains, as determined by a split GFP–luciferase reporter system. The same gB variants were not sufficient to induce syncytia in infected ARPE-19 epithelial cells, suggesting that additional factors are involved. The system described here allows a systematic comparison of the fusogenicity of viral envelope glycoproteins and may help to clarify whether fusion-promoting variants are associated with increased pathogenicity.
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Jeetendra, E., Clinton S. Robison, Lorraine M. Albritton, and Michael A. Whitt. "The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion." Journal of Virology 76, no. 23 (December 1, 2002): 12300–12311. http://dx.doi.org/10.1128/jvi.76.23.12300-12311.2002.
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ABSTRACT Recently we showed that the membrane-proximal stem region of the vesicular stomatitis virus (VSV) G protein ectodomain (G stem [GS]), together with the transmembrane and cytoplasmic domains, was sufficient to mediate efficient VSV budding (C. S. Robison and M. A. Whitt, J. Virol. 74:2239-2246, 2000). Here, we show that GS can also potentiate the membrane fusion activity of heterologous viral fusion proteins when GS is coexpressed with those proteins. For some fusion proteins, there was as much as a 40-fold increase in syncytium formation when GS was coexpressed compared to that seen when the fusion protein was expressed alone. Fusion potentiation by GS was not protein specific, since it occurred with both pH-dependent as well as pH-independent fusion proteins. Using a recombinant vesicular stomatitis virus encoding GS that contained an N-terminal hemagglutinin (HA) tag (GSHA virus), we found that the GSHA virus bound to cells as well as the wild-type virus did at pH 7.0; however, the GSHA virus was noninfectious. Analysis of cells expressing GSHA in a three-color membrane fusion assay revealed that GSHA could induce lipid mixing but not cytoplasmic mixing, indicating that GS can induce hemifusion. Treatment of GSHA virus-bound cells with the membrane-destabilizing drug chlorpromazine rescued the hemifusion block and allowed entry and subsequent replication of GSHA virus, demonstrating that GS-mediated hemifusion was a functional intermediate in the membrane fusion pathway. Using a series of truncation mutants, we also determined that only 14 residues of GS, together with the VSV G transmembrane and cytoplasmic tail, were sufficient for fusion potentiation. To our knowledge, this is the first report which shows that a small domain of one viral glycoprotein can promote the fusion activity of other, unrelated viral glycoproteins.
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Zelus, Bruce D., Jeanne H. Schickli, Dianna M. Blau, Susan R. Weiss, and Kathryn V. Holmes. "Conformational Changes in the Spike Glycoprotein of Murine Coronavirus Are Induced at 37°C either by Soluble Murine CEACAM1 Receptors or by pH 8." Journal of Virology 77, no. 2 (January 15, 2003): 830–40. http://dx.doi.org/10.1128/jvi.77.2.830-840.2003.
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ABSTRACT The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37°C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37°C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4°C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37°C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37°C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered SA59H716D virions with trypsin at 4°C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37°C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.
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Klupp, Barbara, Jan Altenschmidt, Harald Granzow, Walter Fuchs, and Thomas C. Mettenleiter. "Glycoproteins Required for Entry Are Not Necessary for Egress of Pseudorabies Virus." Journal of Virology 82, no. 13 (April 16, 2008): 6299–309. http://dx.doi.org/10.1128/jvi.00386-08.
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ABSTRACT In the current perception of the herpesvirus replication cycle, two fusion processes are thought to occur during entry and nuclear egress. For penetration, glycoproteins gB and gH/gL have been shown to be essential, whereas a possible role of these glycoproteins in nuclear egress remains unclear. Viral envelope glycoproteins have been detected by immunolabeling in the nuclear membrane as well as in primary enveloped particles in several herpesviruses, indicating that they might be involved in the fusion process. Moreover, a herpes simplex virus type 1 mutant simultaneously lacking gB and gH was described to be deficient in nuclear egress (A. Farnsworth, T. W. Wisner, M. Webb, R. Roller, G. Cohen, R. Eisenberg, and D. C. Johnson, Proc. Natl. Acad. Sci. USA 104:10187-10192, 2007). To analyze the situation in the related alphaherpesvirus pseudorabies virus (PrV), mutants carrying single and double deletions of glycoproteins gB, gD, gH, and gL were constructed and characterized. We show here that the simultaneous deletion of gB and gD, gB and gH, gD and gH, or gH and gL has no detectable effect on PrV egress, implying that none of these glycoproteins either singly or in the tested combinations is required for nuclear egress. In addition, immunolabeling studies using different mono- or polyclonal sera raised against various PrV glycoproteins did not reveal the presence of viral glycoproteins in the inner nuclear membrane or in primary virions. Thus, our data strongly suggest that different fusion mechanisms are active during virus entry and egress.
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Eisfeld, Hannah S., Alexander Simonis, Sandra Winter, Jason Chhen, Luisa J. Ströh, Thomas Krey, Manuel Koch, Sebastian J. Theobald, and Jan Rybniker. "Viral Glycoproteins Induce NLRP3 Inflammasome Activation and Pyroptosis in Macrophages." Viruses 13, no. 10 (October 15, 2021): 2076. http://dx.doi.org/10.3390/v13102076.
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Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1β. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1β release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.
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Brighty, David W., and Sushma R. Jassal. "The Synthetic Peptide P-197 Inhibits Human T-Cell Leukemia Virus Type 1 Envelope-Mediated Syncytium Formation by a Mechanism That Is Independent of Hsc70." Journal of Virology 75, no. 21 (November 1, 2001): 10472–78. http://dx.doi.org/10.1128/jvi.75.21.10472-10478.2001.
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ABSTRACT Entry of human T-cell leukemia virus type 1 (HTLV-1) into cells is mediated by the viral envelope glycoproteins gp46 and gp21. The gp46 surface glycoprotein binds to a poorly characterized cell surface receptor, thereby promoting the gp21-dependent fusion of the viral and cellular membranes. Interestingly, a synthetic peptide (P-197) simulating amino acids 197 to 216 of gp46 strongly inhibits envelope-dependent membrane fusion with Molt-4 target cells. It has been suggested that this peptide acts by competitively binding to Hsc70, a putative cellular receptor for HTLV-1. We now demonstrate that P-197 inhibits membrane fusion among diverse HTLV-1-permissive target cells. Importantly, most of these cells lack detectable levels of Hsc70, indicating that P-197 inhibits membrane fusion by a mechanism that is Hsc70 independent. We now suggest that competition for primary receptor binding is unlikely to account for the inhibitory activity of P-197. Understanding the mechanism by which P-197 functions may reveal concepts of general relevance to antiretroviral chemotherapy.