Anti-Chikungunya Virus Monoclonal Antibody That Inhibits Viral Fusion and Release

ABSTRACT Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae. Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a β-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-μm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection. IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

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Effects of an In-Frame Deletion of the6kGene Locus from the Genome of Ross River Virus

Journal of Virology ◽

10.1128/jvi.03192-15 ◽

2016 ◽

Vol 90 (8) ◽

pp. 4150-4159 ◽

Cited By ~ 22

Author(s):

Adam Taylor ◽

Julian V. Melton ◽

Lara J. Herrero ◽

Bastian Thaa ◽

Liis Karo-Astover ◽

...

Keyword(s):

Deletion Mutant ◽

Chikungunya Virus ◽

Vaccine Development ◽

Wild Type Virus ◽

Ross River Virus ◽

Wild Type ◽

Virion Release ◽

Viral Vaccine ◽

Infected Cells ◽

Viral Titers

ABSTRACTThe alphaviral6kgene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the6kproteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel6kin-frame deletion mutant. Comprehensive microscopic analysis revealed that the6kproteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the6kproteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the6kdeletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequentin vivostudies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the6kproteins may contribute to alphaviral disease manifestations and suggest that manipulation of the6kgene may be a potential strategy to facilitate viral vaccine development.IMPORTANCEArthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV6kdeletion mutant. The absence of the6kgene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

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A Conformation-Specific Monoclonal Antibody Reacting with Fusion-Active gp41 from the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

Journal of Virology ◽

10.1128/jvi.72.12.10213-10217.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 10213-10217 ◽

Cited By ~ 122

Author(s):

Shibo Jiang ◽

Kang Lin ◽

Min Lu

Keyword(s):

Monoclonal Antibody ◽

Human Immunodeficiency Virus ◽

Membrane Fusion ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Envelope Glycoprotein ◽

Structural Domain ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays a major role in the membrane fusion step of viral infection. The ectodomain of gp41 contains a six-helix structural domain that likely represents the core of the fusion-active conformation of the molecule. A monoclonal antibody (MAb), designated NC-1, was generated and cloned from a mouse immunized with the model polypeptide N36(L6)C34, which folds into a stable six-helix bundle. NC-1 binds specifically to both the α-helical core domain and the oligomeric forms of gp41. This conformation-dependent reactivity is dramatically reduced by point mutations within the N-terminal coiled-coil region of gp41 which impede formation of the gp41 core. NC-1 binds to the surfaces of HIV-1-infected cells only in the presence of soluble CD4. These results indicate that NC-1 is capable of reacting with fusion-active gp41 in a conformation-specific manner and can be used as a valuable biological reagent for studying the receptor-induced conformational changes in gp41 required for membrane fusion and HIV-1 infection.

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

Journal of Virology ◽

10.1128/jvi.02700-09 ◽

2010 ◽

Vol 84 (12) ◽

pp. 6119-6129 ◽

Cited By ~ 22

Author(s):

Joanne York ◽

Jody D. Berry ◽

Ute Ströher ◽

Qunnu Li ◽

Heinz Feldmann ◽

...

Keyword(s):

Cell Membrane ◽

Membrane Fusion ◽

Host Cell ◽

Envelope Glycoprotein ◽

Fusion Peptide ◽

Intermediate Form ◽

Viral Fusion ◽

Virus Envelope ◽

Host Cell Membrane ◽

Helix Bundle

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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Experimental Evolution To Isolate Vaccinia Virus Adaptive G9 Mutants That Overcome Membrane Fusion Inhibition via the Vaccinia Virus A56/K2 Protein Complex

Journal of Virology ◽

10.1128/jvi.00093-20 ◽

2020 ◽

Vol 94 (10) ◽

Author(s):

Guan-Ci Hong ◽

Chi-Hang Tsai ◽

Wen Chang

Keyword(s):

Vaccinia Virus ◽

Membrane Fusion ◽

Hela Cells ◽

Protein Complex ◽

Experimental Evolution ◽

Low Ph ◽

Mutant Protein ◽

Viral Fusion ◽

Fusion Inhibition ◽

Infected Cells

ABSTRACT For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the ΔA56 virus, the G9H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion. IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter’s G9 subcomponent. Although the G9H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.

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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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