ENTEROVIRUS RECOMBINATION AS A TARGET FOR VIRAL ATTENUATION AND ANTIVIRAL THERAPEUTICS

Kim, Hyejeong

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February 3, 2023

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Kim, Hyejeong
- Affiliation: School of Medicine, Department of Microbiology and Immunology

Abstract

Novel positive-strand RNA viruses continue to emerge without warning, causing epidemics and pandemics. The recent pandemic caused by circulation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has clearly demonstrated the need for universally applicable strategies to eradicate infections and contain outbreaks. As all positive-strand RNA viruses require an RNA-dependent RNA polymerase (RdRp) for genome replication, this conserved enzyme has been a longstanding target for enzyme-mechanism-based strategies for viral attenuation and development of antiviral therapeutics. While much is known about the structure, dynamics, and mechanism of the RdRp, far less is known about RdRp-catalyzed RNA recombination, which results in the creation of recombinant genomes between co-infected viruses.In this dissertation, we use poliovirus (PV) as a primary model system to study RNA recombination as a target for viral attenuation and therapeutic intervention. In Chapter 2, we investigate biochemical properties of the RdRp that can be tuned to modulate the efficiency and/or mechanism of RNA recombination by characterizing derivatives of a PV RdRp mutant, K359H, of which speed and fidelity were perturbed by changing an RdRp active-site residue. Together with a cell-based approach and two distinct biochemical assays for PV recombination, we demonstrated RdRp-mediated template switching as the primary mechanism of copy-choice recombination. Furthermore, our data suggests the existence of additional biochemical properties of the RdRp, rather than the commonly cited properties of speed and fidelity, that are essential for recombination.In Chapter 3, we report the discovery of IRES-independent translation of enterovirus genome. In addition to the genetic evidence, we also demonstrated indirect detection of PV 3CD produced by the IRES-independent mechanism. Moreover, we also demonstrated the non-canonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation.For the RdRp-catalyzed template-switching mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. In Chapter 4, we present our future directions and show preliminary data to investigate how a viral genome is trafficked to the site of genome replication and how recombination could occur.

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