Year of Award

2009

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Other Degree Name/Area of Focus

Molecular Biology

Department or School/College

Division of Biological Sciences

Committee Chair

Jack H. Nunberg

Commitee Members

Stephen J. Lodmell, Mark L. Grimes, Michele A. McGuirl, David J. Poulsen

Keywords

Arenaviruses, Envelope Glycoprotein, Hemorrhagic Fever, Intracellular Trafficking, Stable Signal Peptide, Virus Assembly

Publisher

University of Montana

Abstract

An unusual feature in the arenavirus envelope glycoprotein complex (GP-C) is the presence of a myristoylated stable signal peptide (SSP) in addition to the receptor binding subunit G1 and the transmembrane fusion subunit G2. Genetic studies were employed to understand the structure-function of the GP-C complex, with emphasis on elucidating the role of SSP in arenavirus life cycle. We present genetic evidence that support the inclusion of G2 as a member of Class I viral fusion protein, where membrane fusion is mediated by six-helix bundle structure as in HIV and Influenza viruses. Furthermore, we have identified crucial roles for SSP in the GP-C complex. In the mature glycoprotein complex, SSP assumes a bitopic membrane topology with both its N and C termini in the cytosol, and a short ectodomain loop. This membrane orientation allows it to mask the endogenous endoplasmic reticulum retrieval signals in the cytoplasmic tail of G2 thereby allowing the transit of fully assembled GP-C complex through the Golgi to the cell surface. SSP also interacts with the ectodomain of G2 on the outer surface of the membrane. This interaction is critical in modulating the pH at which the membrane fusion is activated. The SSP-G2 pocket has been identified as the target of newly discovered small-molecule inhibitors of arenaviral entry. Beyond its role in intracellular trafficking and pH-dependent membrane fusion of GP-C complex, SSP might be involved in virus assembly and budding. Studies employing immunogold electron microscopy indicated that GP-C complex clusters into microdomains of 120 nm size independent of other viral proteins. Clustering of GP-C into membrane microdomains is neither influenced by SSP myristoylation nor by the co-expression of the matrix protein do Z. Regions of plasma membrane containing Z not co-localize with GP-C containing microdomains. Clustering of proteins or lipids on the plasma membrane may bring Z and GP-C together at the virus budding sites. Taken together, these data have contributed to the understanding of the unique subunit organization in GP-C complex and the mechanisms underlying efficient co-ordination of these subunits to execute significant functions in the arenavirus life cycle.

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© Copyright 2009 Sudhakar Srinivasarao Agnihothram