Functional and structural studies of Chikungunya Virus nsP1 Protein

Abstract

Chikungunya virus (CHIKV) is a single-stranded, positive-sense RNA virus of the Alphavirus genus. CHIKV is an arbovirus, whose spread is mediated by Aedes species mosquitos and is associated with debilitating joint pain and febrile symptoms in infected humans. A lack of vaccine or specific antivirals, combined with increasing global spread, has facilitated the re-emergence of CHIKV in recent years. Our research focuses on the CHIKV encoded non-structural protein-1 (nsP1), which has methyltransferase activity and is essential for virus genome replication. Through a combination of structural, biochemical and reverse genetic approaches, we aim to investigate the relationship between the molecular structure and both canonical and non-canonical functions of nsP1, at different stages of CHIKV replication. We demonstrated that substitution of an in-frame methionine (M24), towards the N-terminal of nsP1, severely inhibits CHIKV replication in a host cell-dependent manner. Specifically, we demonstrated that an M24>A substitution had no significant effect on sub-genomic replicon replication but blocks production of infectious CHIKV virions – suggesting a role in later stages of virus replication, such as packaging or egress, rather than genome replication or translation. Utilising such a reverse genetic approach, analysis of a panel of M24 substitutions has improved our understanding on non-canonical yet essential functions of nsP1 during CHIKV replication. In order to further elucidate both the structural and biochemical basis for the observed mutant phenotypes, we developed a system for bacterial expression and purification of recombinant CHIKV nsP1 and have established crystallography trails, prior to analysis of the molecular structure by X-ray crystallography.