Browsing by Author "Gyanwali, Rashwita"

Semliki Forest virus (SFV) is a well-studied model virus of medically important mosquito-borne alphaviruses, like chikungunya virus and Sindbis virus. SFV replicates within membrane invaginations called spherules at the host plasma membrane, facilitated by the virus-encoded nsP1 protein. The objectives of this MSc thesis were to identify candidate host proteins interacting with nsP1 based on previous proteomics work and develop a screening workflow to identify stable nsP1 interactors. The overarching aim is to improve our understanding of the roles of host proteins in spherule formation and replication complex functions. The screening workflow involved knockdown assays to assess the antiviral and proviral effects of potential host interactors, followed by cell viability assays for toxicity assessment. Selected promising hits were further investigated for protein interaction with nsP1, which required cloning for mammalian expression and tagging with Myc epitope. Knockdowns resulted in several statistically significantly proviral and antiviral host factors, and all the knockdowns were non-toxic to the cells. Among the significant proviral hits, four promising candidate host proteins were cloned and expressed. Pull-down assays did not reveal stable interactions, suggesting transient or indirect interactions between these host proteins and nsP1. The lack of co-localisation with SFV replication complex supported this finding. This work sheds light on possible transient or indirect interactors of nsP1. The screening workflow effectively identified protein-protein interactions and can be applied to screen additional proteins. Future studies should employ methods suitable for studying transient interactors to gain further insights. This would enhance our understanding of key host proteins in SFV spherule formation and replication, potentially leading to novel antiviral therapies targeting alphavirus replication.