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Journal of Virology, April 2007, p. 4331-4337, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02779-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Bitopic Membrane Topology of the Stable Signal Peptide in the Tripartite Junín Virus GP-C Envelope Glycoprotein Complex{triangledown}

Sudhakar S. Agnihothram,1,2 Joanne York,1 Meg Trahey,1 and Jack H. Nunberg1*

Montana Biotechnology Center,1 Division of Biological Sciences, The University of Montana, Missoula, Montana 598122

Received 15 December 2006/ Accepted 23 January 2007

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 (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.

* Corresponding author. Mailing address: Montana Biotechnology Center, The University of Montana, Science Complex, Room 221, Missoula, MT 59812. Phone: (406) 243-6421. Fax: (406) 243-6425. E-mail: jack.nunberg{at}umontana.edu

{triangledown} Published ahead of print on 31 January 2007.

Journal of Virology, April 2007, p. 4331-4337, Vol. 81, No. 8
0022-538X/07/$08.00+0     doi:10.1128/JVI.02779-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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