Ewing Sarcoma Breakpoint Region 1 (EWSR1) protein binds to the non-structural protein-2 and the non-coding regions of the Chikungunya virus genome to inhibit the virus replication

Chikungunya virus (CHIKV) is an alphavirus with a single-stranded RNA genome. Conserved sequence elements within the 5'- and 3'-non-coding regions (NCRs) of the CHIKV genome interact with host proteins to regulate viral RNA replication. Electrophoretic mobility shift assays (EMSA) and mass spectrometry identified several host RNA-binding proteins that associate with these conserved NCR elements, among which EWSR1 uniquely bound the 3'-ends of both the plus-sense genomic RNA and the minus-sense replication intermediate RNA. EMSA and biolayer interferometry demonstrated that EWSR1 binds strongly to two RNA elements: a 48-nucleotide region in the plus-strand 3'-NCR (3NCR48) and a 50-nucleotide region at the 3'-end of the minus-strand replication intermediate (5NCR-RC50), which is complementary to the genomes 5'-NCR and its adjacent sequence. RNA immunoprecipitation from CHIKV-infected cells confirmed that EWSR1 binds CHIKV RNA in vivo. Functional analyses revealed that CHIKV infection downregulates EWSR1 expression and causes its cytoplasmic accumulation. Knockdown or knockout of EWSR1 expression increased CHIKV genomic RNA levels and viral titers, whereas its overexpression suppressed both. EWSR1 reduced the synthesis of both plus- and minus-sense CHIKV RNAs in infected cells. Additionally, EWSR1 was found to bind CHIKV nsP2 and inhibit its helicase activity, which is critical for viral genome replication. The antiviral activity of EWSR1 extended to Ross River virus but not to Sindbis virus, consistent with conservation of the 48-nucleotide sequence in their respective 3'-NCRs. Together, these findings identify EWSR1 as a novel host restriction factor that limits CHIKV replication through specific interactions with viral RNA and the nsP2 protein. IMPORTANCEChikungunya virus (CHIKV) poses a significant medical challenge, necessitating the development of novel antiviral strategies. This study demonstrates that the host protein EWSR1 plays a key role in controlling CHIKV replication. By binding to specific regions of viral RNA and interacting with nsP2, a critical viral enzyme, EWSR1 inhibits viral RNA synthesis and reduces viral titers. In the absence of EWSR1, CHIKV replicates more efficiently, underscoring its antiviral function. These findings establish EWSR1 as a novel host restriction factor that limits CHIKV replication. Understanding this interaction enhances our knowledge of host-virus dynamics and may guide the design of new antiviral approaches against CHIKV and related alphaviruses.