Journal
JOURNAL OF VIROLOGY
Volume 81, Issue 8, Pages 4331-4337Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.02779-06
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Funding
- NCRR NIH HHS [P20 RR015583, RR01760, RR15583] Funding Source: Medline
- NIAID NIH HHS [U54 AI065357-01, R21 AI059355, R01 AI074818, U54 AI065357, AI059355] Funding Source: Medline
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The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junin 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 (h phi 1 and h phi 2) 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 h phi 2 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.
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