Date of Award

12-16-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Richard Plemper

Second Advisor

Margo Brinton

Third Advisor

Sang-Moo Kang

Abstract

Measles virus (MeV) is considered one of the most contagious human viruses and has recently been declared endemic again in several countries despite a highly efficacious vaccine. The viral RNA dependent RNA polymerase (RdRP) is a heterologous complex comprised of the large protein (L), which provides all catalytic domains for RNA synthesis, and the phosphoprotein (P), which provides chaperone support for L and mediates the interaction between L and the ribonucleoprotein complex (RNP). Though essential for polymerase function, the interface between P and L remains poorly characterized, as well as the role of P in RdRP advancement along the RNP. Through biochemical interface mapping, functional assays, and domain swapping of P, we have identified a bipartite L binding domain on P. One domain consists of a conserved helical motif upstream of the oligomerization domain (OD), and the second is a face of the P X-domain (PXD). Using stoichiometrically-controlled trans-complementation studies and applied mathematical modeling, we also determined the PXD:L interaction to be mutually exclusive to the PXD:N interaction. These findings suggest a model that centers PXD as a master regulator of RdRP advancement.

Rabies virus (RABV) causes a severe and 100% fatal neurological disease that is vaccine preventable and treatable prior to the onset of clinical symptoms. The post-exposure prophylaxis (PEP) for RABV treatment is prohibitively expensive, especially in developing countries where the majority of cases occur and does not confer cross-protection against the newly emerging phylogroup II lyssaviruses. To address the unmet clinical need for cross-protective anti-RABV therapeutics, we developed and implemented an innovative high-throughput screening approach utilizing a novel single cycle RABV reporter strain maintained in BSL-2 laboratory conditions. From our extensive screening library, we have identified the first direct-acting multi-strain RABV entry inhibitor, GRP-60367. Resistance profiling of GRP-60367 revealed escape mutations that accelerate the fusion kinetic of the RABV glycoprotein (G). We have solved two of the feasibility issues with current RABV antiviral drug discovery: i) BSL-2+RABV containment restraints and ii) reliable drug efficacy determination, thus paving the way for future drug discovery campaigns to alleviate the deficit and cost of current therapeutic options against lyssaviruses.

DOI

https://doi.org/10.57709/15901134

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