Date of Award
Doctor of Philosophy (PhD)
Since its emergence in late 2019, the COVID-19 pandemic continues to devastate communities worldwide. Despite the successes in both identifying new therapeutics and immunization strategies that have alleviated the toll on livelihoods, the threat of novel coronavirus (CoV) variants triggering future pandemics necessitates research into the lifecycle of the causative agent, SARS-CoV-2, and more broadly related CoVs. An essential aspect of viral propagation is the replication process in which viral enzymes orchestrate the production of nascent viral RNA genomes. CoV RNA synthesis is mediated by the RNA-dependent RNA polymerase (RdRp) which requires a coterie of viral nucleic acid-metabolizing enzymes to maintain the integrity of the genome. The RdRp and other viral replication enzymes are postulated to interact as part of a large assembly known as the replication-transcription complex (RTC). In chapter II, I explore the basis by which RTC components may interact and reveal coupling of the essential viral helicase, non-structural protein (nsp) 13, with the RdRp in which two copies of nsp13 associate with the holoenzyme-RdRp complex bound to its RNA substrate (referred to as the nsp132-RTC) using cryo-electron microscopy (cryo-EM). Intriguingly, we observed that one of the bound helicases is orientated with an opposing polarity to the RdRp on the template RNA (t-RNA) strand. In chapter III, we investigated the effect of this configuration on the RdRp and identified that nsp13 precipitates RdRp backtracking, which is a universal regulatory feature of many nucleic acid polymerases that describes their reverse motion on the nucleic acid template. The backtracking observation prompted questions as to how the polymerase can elongate RNA in the presence of the helicase. To investigate this, we performed further structural analysis of the nsp132-RTC complex using a combination of single particle cryo-electron microscopy and molecular dynamics (MD) simulation analysis. Our results identified several distinct conformational states of the nsp13 helicase and suggested a mechanism for the nsp132-RTC to turn backtracking on and off; allosterically orchestrating either RNA synthesis or backtracking in response to stimuli at the RdRp active site. As the critical element to viral replication, the RTC is a target for clinically used antivirals such as remdesivir and molnupiravir. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA, while antiviral nucleoside analogs must compete with the natural NTPs to be effective. How the SARS-CoV-2 RTC discriminates between the natural NTPs, and how antiviral nucleoside analogs compete, has not been discerned in detail. Therefore, I used cryo-electron microscopy to visualize the RTC bound to each of the natural NTPs in states poised for incorporation in chapter IV. Furthermore, I investigated the RTC with the active metabolite of remdesivir, remdesivir triphosphate (RDV-TP), highlighting the structural basis for the selective incorporation of RDV-TP over its natural counterpart ATP. My results elucidate the suite of interactions required for NTP recognition, informing the rational design of antivirals. To further our understanding on remdesivir’s mechanism of action, I analyzed the effect of remdesivir when incorporated in the newly replicated viral RNA to establish how it may impede viral RNA synthesis following treatment (template-dependent inhibition). My structural studies illustrate how remdesivir perturbs nucleotide incorporation by stalling the polymerase active site in a catalytically unfavorable state. In the final chapter, I explore the implications of our investigations and how they may spur further studies on the underlying mechanisms of CoV RNA synthesis. In particular, I will discuss how the helicase may be involved in the process of template switching required to produce subgenomic-mRNAs (sg-mRNAs) during ‘transcription’ as well as how helicase induced backtracking may promote replication coupled repair. Furthermore, I will conclude by describing the remaining gaps in our knowledge to help guide new research on the CoV replication cycle.
Malone, Brandon F., "Structures, Functions, and Druggability of the SARS-CoV-2 Replication–Transcription Complex" (2023). Student Theses and Dissertations. 722.
Available for download on Wednesday, January 31, 2024