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

2007 ◽  
Vol 81 (24) ◽  
pp. 13385-13391 ◽  
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.

scholarly journals The Signal Peptide of the Junín Arenavirus Envelope Glycoprotein Is Myristoylated and Forms an Essential Subunit of the Mature G1-G2 Complex

2004 ◽  
Vol 78 (19) ◽  
pp. 10783-10792 ◽  
Author(s):  
Joanne York ◽  
Victor Romanowski ◽  
Min Lu ◽  
Jack H. Nunberg
Keyword(s):  
Signal Peptide ◽  
Envelope Glycoprotein ◽  
Hemorrhagic Fever ◽  
Gene Replacement ◽  
Etiologic Agent ◽  
Virus Envelope ◽  
Glycoprotein Complex ◽  
Amino Acid Signal Peptide ◽  
Dependent Cell ◽  
And Function

ABSTRACT Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking.

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

2010 ◽  
Vol 84 (12) ◽  
pp. 6119-6129 ◽  
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.

scholarly journals Role of the Stable Signal Peptide of Junín Arenavirus Envelope Glycoprotein in pH-Dependent Membrane Fusion

2006 ◽  
Vol 80 (15) ◽  
pp. 7775-7780 ◽  
Author(s):  
Joanne York ◽  
Jack H. Nunberg
Keyword(s):  
Amino Acid ◽  
Membrane Fusion ◽  
Signal Peptide ◽  
Envelope Glycoprotein ◽  
Amino Acid Signal Peptide ◽  
Ph Dependent ◽  
Mediate Cell ◽  
C Complex ◽  
Stable Signal

ABSTRACT The envelope glycoprotein of the arenaviruses (GP-C) is unusual in that the mature complex retains the cleaved, 58-amino-acid signal peptide. Association of this stable signal peptide (SSP) has been shown to be essential for intracellular trafficking and proteolytic maturation of the GP-C complex. We identify here a specific and previously unrecognized role of SSP in pH-dependent membrane fusion. Amino acid substitutions that alter the positive charge at lysine K33 in SSP affect the ability of GP-C to mediate cell-cell fusion and the threshold pH at which membrane fusion is triggered. Based on the presumed location of K33 at or near the luminal domain of SSP, we postulate that SSP interacts with the membrane-proximal or transmembrane regions of the G2 fusion protein. This unique organization of the GP-C complex may suggest novel strategies for intervention in arenavirus infection.

scholarly journals Structure of a Zinc-binding Domain in the Junín Virus Envelope Glycoprotein

2010 ◽  
Vol 286 (2) ◽  
pp. 1528-1536 ◽  
Author(s):  
Klára Briknarová ◽  
Celestine J. Thomas ◽  
Joanne York ◽  
Jack H. Nunberg
Keyword(s):  
Envelope Glycoprotein ◽  
Binding Domain ◽  
Zinc Binding ◽  
Virus Envelope ◽  
Junin Virus ◽  
Zinc Binding Domain ◽  
Junín Virus

scholarly journals Probing the antigenicity of hepatitis C virus envelope glycoprotein complex by high-throughput mutagenesis

2017 ◽  
Vol 13 (12) ◽  
pp. e1006735 ◽  
Author(s):  
Radhika Gopal ◽  
Kelli Jackson ◽  
Netanel Tzarum ◽  
Leopold Kong ◽  
Andrew Ettenger ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
Envelope Glycoprotein ◽  
Virus Envelope ◽  
Glycoprotein Complex

scholarly journals Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Katarzyna Grzyb ◽  
Anna Czarnota ◽  
Agnieszka Brzozowska ◽  
Anna Cieślik ◽  
Łukasz Rąbalski ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Envelope Glycoprotein ◽  
Functional Characterization ◽  
Virus Envelope ◽  
Glycoprotein Complex

scholarly journals Cytolysis by CCR5-Using Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Is Dependent on Membrane Fusion and Can Be Inhibited by High Levels of CD4 Expression

2003 ◽  
Vol 77 (12) ◽  
pp. 6645-6659 ◽  
Author(s):  
Jason A. LaBonte ◽  
Navid Madani ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Membrane Fusion ◽  
Human Immunodeficiency Virus Type ◽  
Envelope Glycoprotein ◽  
Envelope Glycoproteins ◽  
Target Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT T-tropic (X4) and dualtropic (R5X4) human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins kill primary and immortalized CD4+ CXCR4+ T cells by mechanisms involving membrane fusion. However, because much of HIV-1 infection in vivo is mediated by M-tropic (R5) viruses whose envelope glycoproteins use CCR5 as a coreceptor, we tested a panel of R5 and R5X4 envelope glycoproteins for their ability to lyse CCR5+ target cells. As is the case for CXCR4+ target cells, HIV-1 envelope glycoproteins expressed by single-round HIV-1 vectors killed transduced CD4+ CCR5+ cells in a membrane fusion-dependent manner. Furthermore, a CD4-independent R5 HIV-1 envelope glycoprotein was able to kill CD4-negative target cells expressing CCR5, demonstrating that CD4 is not intrinsically required for the induction of death. Interestingly, high levels of CD4 expression protected cells from lysis and syncytium formation mediated by the HIV-1 envelope glycoproteins. Immunoprecipitation experiments showed that high levels of CD4 coexpression inhibited proteolytic processing of the HIV-1 envelope glycoprotein precursor gp160. This inhibition could be overcome by decreasing the CD4 binding ability of gp120. Studies were also undertaken to investigate the ability of virion-bound HIV-1 envelope glycoproteins to kill primary CD4+ T cells. However, neither X4 nor R5X4 envelope glycoproteins on noninfectious virions caused death in primary CD4+ T cells. These results demonstrate that the interaction of CCR5 with R5 HIV-1 envelope glycoproteins capable of inducing membrane fusion leads to cell lysis; overexpression of CD4 can inhibit cell killing by limiting envelope glycoprotein processing.

scholarly journals Intersubunit Interactions Modulate pH-Induced Activation of Membrane Fusion by the Junín Virus Envelope Glycoprotein GPC

2009 ◽  
Vol 83 (9) ◽  
pp. 4121-4126 ◽  
Author(s):  
Joanne York ◽  
Jack H. Nunberg
Keyword(s):  
Membrane Fusion ◽  
Envelope Glycoprotein ◽  
Specific Interaction ◽  
Hemorrhagic Fever ◽  
Virus Envelope ◽  
Alanine Scanning Mutagenesis ◽  
Ph Dependent ◽  
Membrane Spanning ◽  
Intersubunit Interactions ◽  
Stable Signal

ABSTRACT The mature arenavirus envelope glycoprotein GPC is a tripartite complex comprising a stable signal peptide (SSP) in addition to the receptor-binding (G1) and transmembrane fusion (G2) subunits. We have shown previously that SSP is a key element in GPC-mediated membrane fusion, and that GPC sensitivity to acidic pH is modulated in part through the lysine residue at position 33 in the ectodomain loop of SSP (J. York and J. H. Nunberg, J. Virol. 80:7775-7780, 2006). A glutamine substitution at this position stabilizes the native GPC complex and thereby prevents the induction of pH-dependent membrane fusion. In efforts to identify the intersubunit interactions of K33, we performed alanine-scanning mutagenesis at charged residues in the membrane-proximal ectodomain of G2 and determined the ability of these mutations to rescue the fusion deficiency in K33Q GPC. Four second-site mutations that specifically complement K33Q were identified (D400A, E410A, R414A, and K417A). Moreover, complementation was also observed at three hydrophobic positions in the membrane-spanning domain of G2 (F427, W428, and F438). Interestingly, all of the complementing mutations restored wild-type pH sensitivity to the K33Q mutant, while none themselves affected the pH of membrane fusion. Our studies demonstrate a specific interaction between SSP and G2 that is involved in priming the native GPC complex for pH-induced membrane fusion. Importantly, this pH-dependent interaction has been shown to be vulnerable to small-molecule compounds that stabilize the native complex and prevent the activation of membrane fusion. A detailed mechanistic understanding of the control of GPC-mediated membrane fusion will be important in guiding the development of effective therapeutics against arenaviral hemorrhagic fever.

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