Inter-domain Flexibility of Chikungunya Virus nsP2 Helicase-Protease Differentially Influences Viral RNA Replication and Infectivity

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for chikungunya fever. Nonstructural protein 2 (nsP2), a multifunctional protein essential for viral replication, has an N-terminal helicase region (nsP2h), which as both nucleotide triphosphatase and RNA triphosphatase activities, as well as a C-terminal cysteine protease region (nsP2p), which is responsible for nonstructural polyprotein processing. The two functional units are connected through a linker of fourteen residues. Although crystal structures of the helicase and protease regions of CHIKV nsP2 have been solved separately, the conformational arrangement of the full-length nsP2 and the biological role of the linker remain elusive. Using the small-angle X-ray scattering (SAXS) method, we demonstrated that the full-length nsP2 is elongated and partially folded in solution. The reconstructed model of the structure of nsP2 contains a flexible inter-domain linker, and there is no direct interaction between the two structured regions. To examine the function of the inter-domain linker, we constructed and characterized a set of CHIKV mutants. The deletion of three or five amino acid residues in the linker region resulted in a modest defect in viral RNA replication and transcription but completely abolished viral infectivity. In contrast, increasing the flexibility of nsP2 by lengthening the inter-domain linker increased both genomic RNA replication and viral infectivity. The enzymatic activities of the corresponding mutant proteins were largely unaffected. This work suggests that increasing the inter-domain flexibility of nsP2 could facilitate the assembly of the replication complex (RC) with increased efficiency and promote virus production.IMPORTANCE: CHIKV nsP2 plays multiple roles in viral RNA replication and virus-host interactions. The helicase and protease regions of nsP2 are connected through a short "linker". Here, we determined that the conformation of full-length CHIKV nsP2 is elongated and that the protein is flexible in solution. ...