An Antibody Directed against the Fusion Peptide of Junín Virus Envelope Glycoprotein GPC Inhibits pH-Induced Membrane Fusion

ABSTRACT The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway. Inhibition of syncytium formation requires that F100G5 be present concomitant with exposure of GPC to acidic pH. We show that F100G5 recognizes neither the six-helix bundle nor the larger trimer-of-hairpins structure in the postfusion form of G2. Rather, Western blot analysis using recombinant proteins and a panel of alanine-scanning GPC mutants revealed that F100G5 binding is dependent on an invariant lysine residue (K283) near the N terminus of G2, in the so-called fusion peptide that inserts into the host cell membrane during the fusion process. The F100G5 epitope is located in the internal segment of the bipartite GPC fusion peptide, which also contains four conserved cysteine residues, raising the possibility that this fusion peptide may be highly structured. Collectively, our studies indicate that F100G5 identifies an on-path intermediate form of GPC. Binding to the transiently exposed fusion peptide may interfere with G2 insertion into the host cell membrane. Strategies to effectively target fusion peptide function in the endosome may lead to novel classes of antiviral agents.

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Exaptation of Retroviral Syncytin for Development of Syncytialized Placenta, Its Limited Homology to the SARS-CoV-2 Spike Protein and Arguments against Disturbing Narrative in the Context of COVID-19 Vaccination

Biology ◽

10.3390/biology10030238 ◽

2021 ◽

Vol 10 (3) ◽

pp. 238

Author(s):

Malgorzata Kloc ◽

Ahmed Uosef ◽

Jacek Z. Kubiak ◽

Rafik M. Ghobrial

Keyword(s):

Cell Membrane ◽

Host Cell ◽

Envelope Glycoprotein ◽

Mammalian Species ◽

Viral Envelope ◽

Spike Protein ◽

Mammalian Evolution ◽

Trophoblast Cells ◽

Host Cell Membrane ◽

Uterine Endometrium

Human placenta formation relies on the interaction between fused trophoblast cells of the embryo with uterine endometrium. The fusion between trophoblast cells, first into cytotrophoblast and then into syncytiotrophoblast, is facilitated by the fusogenic protein syncytin. Syncytin derives from an envelope glycoprotein (ENV) of retroviral origin. In exogenous retroviruses, the envelope glycoproteins coded by env genes allow fusion of the viral envelope with the host cell membrane and entry of the virus into a host cell. During mammalian evolution, the env genes have been repeatedly, and independently, captured by various mammalian species to facilitate the formation of the placenta. Such a shift in the function of a gene, or a trait, for a different purpose during evolution is called an exaptation (co-option). We discuss the structure and origin of the placenta, the fusogenic and non-fusogenic functions of syncytin, and the mechanism of cell fusion. We also comment on an alleged danger of the COVID-19 vaccine based on the presupposed similarity between syncytin and the SARS-CoV-2 spike protein.

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The hr1 and Fusion Peptide Regions of the Subgroup B Avian Sarcoma and Leukosis Virus Envelope Glycoprotein Influence Low pH-Dependent Membrane Fusion

PLoS ONE ◽

10.1371/journal.pone.0000171 ◽

2007 ◽

Vol 2 (1) ◽

pp. e171 ◽

Cited By ~ 8

Author(s):

Angeline Rose Babel ◽

James Bruce ◽

John A.T. Young

Keyword(s):

Membrane Fusion ◽

Envelope Glycoprotein ◽

Fusion Peptide ◽

Low Ph ◽

Virus Envelope ◽

Ph Dependent ◽

Avian Sarcoma

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NMR Structure of the FIV gp36 C-terminal Heptad Repeat and Membrane-Proximal External Region

International Journal of Molecular Sciences ◽

10.3390/ijms21062037 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2037

Author(s):

Manuela Grimaldi ◽

Michela Buonocore ◽

Mario Scrima ◽

Ilaria Stillitano ◽

Gerardino D’Errico ◽

...

Keyword(s):

Cell Membrane ◽

Host Cell ◽

Lipid Membrane ◽

Nmr Structure ◽

Heptad Repeat ◽

Virus Envelope ◽

External Region ◽

Host Cell Membrane ◽

Membrane Proximal External Region ◽

Immunodeficiency Virus

Feline immunodeficiency virus (FIV), a lentivirus causing an immunodeficiency syndrome in cats, represents a relevant model of pre-screening therapies for human immunodeficiency virus (HIV). The envelope glycoproteins gp36 in FIV and gp41 in HIV mediate the fusion of the virus with the host cell membrane. They have a common structural framework in the C-terminal region that includes a Trp-rich membrane-proximal external region (MPER) and a C-terminal heptad repeat (CHR). MPER is essential for the correct positioning of gp36 on the lipid membrane, whereas CHR is essential for the stabilization of the low-energy six-helical bundle (6HB) that is necessary for the fusion of the virus envelope with the cell membrane. Conformational data for gp36 are missing, and several aspects of the MPER structure of different lentiviruses are still debated. In the present work, we report the structural investigation of a gp36 construct that includes the MPER and part of the CHR domain (737-786gp36 CHR–MPER). Using 2D and 3D homo and heteronuclear NMR spectra on 15N and 13C double-labelled samples, we solved the NMR structure in micelles composed of dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) 90/10 M: M. The structure of 737-786gp36 CHR–MPER is characterized by a helix–turn–helix motif, with a regular α-helix and a moderately flexible 310 helix, characterizing the CHR and the MPER domains, respectively. The two helices are linked by a flexible loop regulating their orientation at a ~43° angle. We investigated the positioning of 737-786gp36 CHR–MPER on the lipid membrane using spin label-enhanced NMR and ESR spectroscopies. On a different scale, using confocal microscopy imaging, we studied the effect of 737-786gp36 CHR–MPER on 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPC/DOPG) multilamellar vesicles (MLVs). This effect results in membrane budding and tubulation that is reminiscent of a membrane-plasticizing role that is typical of MPER domains during the event in which the virus envelope merges with the host cell membrane.

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Viral Membrane Fusion and the Transmembrane Domain

Viruses ◽

10.3390/v12070693 ◽

2020 ◽

Vol 12 (7) ◽

pp. 693 ◽

Cited By ~ 1

Author(s):

Chelsea T. Barrett ◽

Rebecca Ellis Dutch

Keyword(s):

Membrane Fusion ◽

Host Cell ◽

Fusion Proteins ◽

Transmembrane Domain ◽

Critical Role ◽

Viral Fusion ◽

Transmembrane Region ◽

Host Cell Membrane ◽

The Past ◽

Viral Fusion Proteins

Initiation of host cell infection by an enveloped virus requires a viral-to-host cell membrane fusion event. This event is mediated by at least one viral transmembrane glycoprotein, termed the fusion protein, which is a key therapeutic target. Viral fusion proteins have been studied for decades, and numerous critical insights into their function have been elucidated. However, the transmembrane region remains one of the most poorly understood facets of these proteins. In the past ten years, the field has made significant advances in understanding the role of the membrane-spanning region of viral fusion proteins. We summarize developments made in the past decade that have contributed to the understanding of the transmembrane region of viral fusion proteins, highlighting not only their critical role in the membrane fusion process, but further demonstrating their involvement in several aspects of the viral lifecycle.

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Entry of an insect poxvirus by fusion of the virus envelope with the host cell membrane

Virology ◽

10.1016/0042-6822(73)90422-4 ◽

1973 ◽

Vol 52 (1) ◽

pp. 305-309 ◽

Cited By ~ 32

Author(s):

Robert R. Granados

Keyword(s):

Cell Membrane ◽

Host Cell ◽

Virus Envelope ◽

Host Cell Membrane

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Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis

Journal of Virology ◽

10.1128/jvi.01124-16 ◽

2016 ◽

Vol 90 (18) ◽

pp. 8341-8350 ◽

Cited By ~ 8

Author(s):

Joanne York ◽

Jack H. Nunberg

Keyword(s):

Public Health ◽

Membrane Fusion ◽

Host Cell ◽

Signal Peptide ◽

Envelope Glycoprotein ◽

Reverse Genetics ◽

Fusion Activity ◽

Virus Envelope ◽

Stable Signal

ABSTRACTArenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ∼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an ∼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes.IMPORTANCEHemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact virion. We find that nonmyristoylated GPC mutants of the Candid #1 strain of Junín virus display a commensurate deficiency in their infectivity, albeit without additional defects in virion assembly and budding. These results suggest that SSP myristoylation may function late in the fusion process to facilitate merging of the viral and cellular membranes. Antiviral agents that target this novel aspect of GPC membrane fusion may be useful in the treatment of arenavirus hemorrhagic fevers.

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Exploitation of the Host Cell Membrane Fusion Machinery by Leishmania Is Part of the Infection Process

PLoS Pathogens ◽

10.1371/journal.ppat.1005962 ◽

2016 ◽

Vol 12 (12) ◽

pp. e1005962 ◽

Cited By ~ 6

Author(s):

Christine Matte ◽

Albert Descoteaux

Keyword(s):

Cell Membrane ◽

Membrane Fusion ◽

Host Cell ◽

Infection Process ◽

Host Cell Membrane

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A Novel Zinc-Binding Domain Is Essential for Formation of the Functional Junín Virus Envelope Glycoprotein Complex

Journal of Virology ◽

10.1128/jvi.01785-07 ◽

2007 ◽

Vol 81 (24) ◽

pp. 13385-13391 ◽

Cited By ~ 38

Author(s):

Joanne York ◽

Jack H. Nunberg

Keyword(s):

Membrane Fusion ◽

Envelope Glycoprotein ◽

Zinc Binding ◽

Target Cells ◽

Viral Fusion ◽

Virus Envelope ◽

Glycoprotein Complex ◽

Amino Acid Signal Peptide ◽

C Complex ◽

Zinc Binding Domain

ABSTRACT The envelope glycoprotein of the Junín arenavirus (GP-C) mediates entry into target cells through a pH-dependent membrane fusion mechanism. Unlike other class I viral fusion proteins, the mature GP-C complex retains a cleaved, 58-amino-acid signal peptide (SSP) as an essential subunit, required both for trafficking of GP-C to the cell surface and for the activation of membrane fusion. SSP has been shown to associate noncovalently in GP-C via the cytoplasmic domain (CTD) of the transmembrane fusion subunit G2. In this report we investigate the molecular basis for this intersubunit interaction. We identify an invariant series of six cysteine and histidine residues in the CTD of G2 that is essential for incorporation of SSP in the GP-C complex. Moreover, we show that a CTD peptide fragment containing His-447, His-449, and Cys-455 specifically binds Zn2+ at subnanomolar concentrations. Together, these results suggest a zinc finger-like domain structure in the CTD of G2. We propose that the remaining residues in the series (His-459, Cys-467, and Cys-469) form an intersubunit zinc-binding center that incorporates Cys-57 of SSP. This unusual motif may act to retain SSP in the GP-C complex and position the ectodomain loop of SSP for its role in modulating membrane fusion activity. The unique tripartite organization of GP-C could provide novel molecular targets for therapeutic intervention in arenaviral disease.

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