Year of Award

2016

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Cellular, Molecular and Microbial Biology

Department or School/College

Division of Biological Sciences

Committee Chair

Jack H. Nunberg

Commitee Members

J. Stephen Lodmell, Brent J. Ryckman, Stephen R. Sprang, Mark A. Pershouse

Keywords

Arenavirus, Glycoprotein complex, GPC, Hemorrhagic fever, inhibition, Membrane fusion

Abstract

Arenaviruses, the causative agents of severe hemorrhagic fevers, are endemic in rodent populations and can be transmitted to humans by contact. Without effective treatment or licensed vaccines, these viruses pose serious public health and biodefense concern. The sole treatment option is the off-label use of the nucleoside analog ribavirin, which is effective only when given at an early stage of infection and shows significant toxicity in humans. Hence, there exists a clear need for developing better therapies. Arenavirus entry into the cell is initiated by the virus envelope glycoprotein complex (GPC), primed to undergo conformational changes triggered by the acidic pH of the maturing endosome, leading to virus and endosomal membrane fusion. Thus, GPC represents as an important molecular target for therapeutic intervention. Recently, several chemically diverse small-molecule fusion inhibitors were identified that block virus entry by stabilizing the prefusion form of GPC against pH-activation. Improved structural and mechanistic understanding of pH-dependent membrane fusion will advance the design and development of potent inhibitors. Here we report that recombinant native-like GPC can be expressed and purified from insect cells, and mediate pH-dependent membrane fusion when reconstituted into proteoliposomes. This fusion reaction is inhibited by small-molecule fusion inhibitors. Further, I show the first physical evidence of binding of small-molecule inhibitors to the pH-sensitive SSP-GP2 interface using photoreactive inhibitors. In addition, I explored mechanism of pH-induced activation of membrane fusion in Old World Lassa virus (LASV). Although the pH-induced activation mechanism is similar to the well-studied New World Junφn virus (JUNV), the differences lie in the usage of an additional secondary (LAMP1) receptor for LASV entry. Another antiviral strategy is to block the packaging and release of virus particles from an infected host cell. Studies have shown that the viral matrix protein Z plays a critical role in virus assembly and budding. Additionally, accumulation of Z at the plasma membrane and interaction with GPC and nucleoprotein (NP) is thought to orchestrate the assembly and budding events. The assembly process involving interaction of GPC with Z has not been clearly understood. I explored the use of confocal microscopy approach to study the association of GPC and Z at the plasma membrane during assembly of the virus.

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© Copyright 2016 SUNDARESH SHANKAR