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.

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