scholarly journals Distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the Junín virus envelope glycoprotein

Virology ◽  
2007 ◽  
Vol 359 (1) ◽  
pp. 72-81 ◽  
Author(s):  
Joanne York ◽  
Jack H. Nunberg
Keyword(s):  
Signal Peptide ◽  
Envelope Glycoprotein ◽  
Signal Peptidase ◽  
Virus Envelope ◽  
Junin Virus ◽  
And Function ◽  
Stable Signal ◽  
Junín Virus

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 Role of the Stable Signal Peptide and Cytoplasmic Domain of G2 in Regulating Intracellular Transport of the Junín Virus Envelope Glycoprotein Complex

2006 ◽  
Vol 80 (11) ◽  
pp. 5189-5198 ◽  
Author(s):  
Sudhakar S. Agnihothram ◽  
Joanne York ◽  
Jack H. Nunberg
Keyword(s):  
Cell Surface ◽  
Signal Peptide ◽  
Envelope Glycoprotein ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Virus Envelope ◽  
Quality Control Process ◽  
C Complex ◽  
Stable Signal

ABSTRACT Enveloped viruses utilize the membranous compartments of the host cell for the assembly and budding of new virion particles. In this report, we have investigated the biogenesis and trafficking of the envelope glycoprotein (GP-C) of the Junín arenavirus. The mature GP-C complex is unusual in that it retains a stable signal peptide (SSP) as an essential component in association with the typical receptor-binding (G1) and transmembrane fusion (G2) subunits. We demonstrate that, in the absence of SSP, the G1-G2 precursor is restricted to the endoplasmic reticulum (ER). This constraint is relieved by coexpression of SSP in trans, allowing transit of the assembled GP-C complex through the Golgi and to the cell surface, the site of arenavirus budding. Transport of a chimeric CD4 glycoprotein bearing the transmembrane and cytoplasmic domains of G2 is similarly regulated by SSP association. Truncations to the cytoplasmic domain of G2 abrogate SSP association yet now permit transport of the G1-G2 precursor to the cell surface. Thus, the cytoplasmic domain of G2 is an important determinant for both ER localization and its control through SSP binding. Alanine mutations to either of two dibasic amino acid motifs in the G2 cytoplasmic domain can also mobilize the G1-G2 precursor for transit through the Golgi. Taken together, our results suggest that SSP binding masks endogenous ER localization signals in the cytoplasmic domain of G2 to ensure that only the fully assembled, tripartite GP-C complex is transported for virion assembly. This quality control process points to an important role of SSP in the structure and function of the arenavirus envelope glycoprotein.

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

2016 ◽  
Vol 90 (18) ◽  
pp. 8341-8350 ◽  
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.

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.

scholarly journals The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain

Virology ◽  
2013 ◽  
Vol 436 (1) ◽  
pp. 127-133 ◽  
Author(s):  
Dominique J. Burri ◽  
Antonella Pasquato ◽  
Joel Ramos da Palma ◽  
Sebastien Igonet ◽  
Michael B.A. Oldstone ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Envelope Glycoprotein ◽  
Proteolytic Processing ◽  
Old World ◽  
Stable Signal

A mouse attenuated mutant of Junin virus with an altered envelope glycoprotein

1990 ◽  
Vol 111 (3-4) ◽  
pp. 257-262 ◽  
Author(s):  
L. A. Scolaro ◽  
Susana E. Mersich ◽  
Elsa B. Damonte
Keyword(s):  
Envelope Glycoprotein ◽  
Junin Virus ◽  
Junín Virus

scholarly journals Bitopic Membrane Topology of the Stable Signal Peptide in the Tripartite Junín Virus GP-C Envelope Glycoprotein Complex

2007 ◽  
Vol 81 (8) ◽  
pp. 4331-4337 ◽  
Author(s):  
Sudhakar S. Agnihothram ◽  
Joanne York ◽  
Meg Trahey ◽  
Jack H. Nunberg
Keyword(s):  
Membrane Fusion ◽  
Signal Peptide ◽  
Envelope Glycoprotein ◽  
Lipid Membrane ◽  
Critical Role ◽  
Membrane Topology ◽  
Single Amino Acid ◽  
Fusion Activity ◽  
C Complex ◽  
Stable Signal

ABSTRACT The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (hφ1 and hφ2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning hφ2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex.

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.

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