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 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 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 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

Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains

1991 ◽  
Vol 11 (5) ◽  
pp. 2675-2685
Author(s):  
A Kundu ◽  
M A Jabbar ◽  
D P Nayak
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Type Ii ◽  
Human Influenza Virus ◽  
Chimeric Genes ◽  
Signal Anchor

We investigated the role of cytoplasmic and anchor domains of type II glycoproteins in intracellular transport, oligomerization, and endocytosis by expressing the wild-type and chimeric genes in mammalian cells. Chimeric genes were constructed by exchanging the DNA segments that encode the cytoplasmic and anchor domains between the human influenza virus (A/WSN/33) neuraminidase (NA) and transferrin receptor (TR). The chimeric proteins in which domains were exchanged precisely were productively targeted to the cell surface. However, the proteins appeared to assemble differently in the intracellular compartment. For example, while TR existed predominantly as a dimer, NATR delta 90, containing the cytoplasmic and signal-anchor domains of NA and the ectodomain of TR, was present as a tetramer, a dimer, and a monomer. Similarly, the influenza virus NA existed predominantly as a tetramer but TRNA delta 35, in which the cytoplasmic and signal-anchor domains of TR were joined to the ectodomain of NA, existed predominantly as a dimer, suggesting that the cytoplasmic and anchor domains of type II glycoproteins affect the subunit assembly of heterologous ectodomains. In addition, we analyzed the role of the cytoplasmic domain in endocytosis. NA and NATR delta 90 did not undergo endocytosis, whereas both TR and TRNA delta 35 were internalized efficiently, demonstrating that the NH2 cytoplasmic domain of TR was capable of internalizing a heterologous ectodomain (NA) from the cell surface.

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 Cell surface transport, oligomerization, and endocytosis of chimeric type II glycoproteins: role of cytoplasmic and anchor domains.

1991 ◽  
Vol 11 (5) ◽  
pp. 2675-2685 ◽  
Author(s):  
A Kundu ◽  
M A Jabbar ◽  
D P Nayak
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Cytoplasmic Domain ◽  
Type Ii ◽  
Human Influenza Virus ◽  
Chimeric Genes ◽  
Signal Anchor

We investigated the role of cytoplasmic and anchor domains of type II glycoproteins in intracellular transport, oligomerization, and endocytosis by expressing the wild-type and chimeric genes in mammalian cells. Chimeric genes were constructed by exchanging the DNA segments that encode the cytoplasmic and anchor domains between the human influenza virus (A/WSN/33) neuraminidase (NA) and transferrin receptor (TR). The chimeric proteins in which domains were exchanged precisely were productively targeted to the cell surface. However, the proteins appeared to assemble differently in the intracellular compartment. For example, while TR existed predominantly as a dimer, NATR delta 90, containing the cytoplasmic and signal-anchor domains of NA and the ectodomain of TR, was present as a tetramer, a dimer, and a monomer. Similarly, the influenza virus NA existed predominantly as a tetramer but TRNA delta 35, in which the cytoplasmic and signal-anchor domains of TR were joined to the ectodomain of NA, existed predominantly as a dimer, suggesting that the cytoplasmic and anchor domains of type II glycoproteins affect the subunit assembly of heterologous ectodomains. In addition, we analyzed the role of the cytoplasmic domain in endocytosis. NA and NATR delta 90 did not undergo endocytosis, whereas both TR and TRNA delta 35 were internalized efficiently, demonstrating that the NH2 cytoplasmic domain of TR was capable of internalizing a heterologous ectodomain (NA) from the cell surface.

scholarly journals Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11

1994 ◽  
Vol 302 (2) ◽  
pp. 451-454 ◽  
Author(s):  
M H Lafrance ◽  
C Vézina ◽  
Q Wang ◽  
G Boileau ◽  
P Crine ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Neutral Endopeptidase ◽  
Soluble Form ◽  
Cell Extracts ◽  
Glycosylation Sites ◽  
Sugar Addition ◽  
Membrane Bound

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

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