Characterizing the alphavirus replication complex to develop antivirals and next generation self-amplifying RNA based therapeutics

• Main Author: Law, Michelle Cheok Yien
• Other Authors: Franklin Zhong Lei
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2024
• Subjects: Medicine, Health and Life Sciences; RNA virus; Alphavirus; Self-amplifying RNA; Antivirals; 5' cap
• Online Access: https://hdl.handle.net/10356/180674
• Institution: Nanyang Technological University

Alphaviruses such as chikungunya virus and Venezuelan equine encephalitis virus are medically relevant pathogens which can affect human health and disease with no approved therapies. They are enveloped viruses which contain a single stranded positive sense RNA genome. The genome is approximately 11-12 kb containing two reading frames. The first open reading frame encodes the nonstructural proteins which assemble into the replication complex for positive and negative strand RNA synthesis. The alphavirus replication complex and its components serve as ideal targets for antiviral drug development due to their essential role in the virus life cycle. Simultaneously, its serves as a potential platform for new vaccines and therapies in self-amplifying RNAs (saRNA). Hence, this thesis reports on their two complimentary research arms centered on the alphavirus replication complex and its components. First, it characterizes nonstructural protein 1 in alphavirus genome capping, its impact on virus-host interaction and role in viral replication. This work systematically dissects the capping activity, revealing its ability to recognize 5′-end AU sequences in RNA and demonstrating its reversible capping reaction. The research also highlights nsP1’s potential involvement in activating the type I interferon (IFN) immune response through decapping activity mediated through RIG-I. This work provides insights into a novel mechanism for alphavirus-induced antiviral response. Next, it examines the potential of targeting nonstructural protein 1 and the alphavirus replication complex for antiviral development. Compounds like 6′-β-Fluoro-homoaristeromycin (FHA) and MADTP series exhibit promising inhibition of CHIKV replication by targeting nsP1 at different sites. Cryogenic electron microscopy structures reveal FHA targeting the SAM binding site while MADTP functioning like a GTP analogue. Drugs targeting the replication complex have been developed for other alphaviruses such as Venezuelan Equine Encephalitis Virus and applying a similar strategy for CHIKV has shown promise, showcasing potential leads for antiviral drug development. Finally, the thesis explores introducing modifications to improve cellular translation for the development of alphavirus based saRNAs. The study delves into the detailed analysis of Venezuelan equine encephalitis virus (VEEV) saRNAs and a novel rubella virus (RuV) saRNAs, uncovering their potential in therapeutic and vaccine development. The work emphasizes the need for further refinement in saRNA vaccine design, contributing valuable insights for advancing RNA therapeutics and overcoming existing challenges in saRNA-based approaches.