ARRDC1 inhibits the replication of Semliki Forest virus by regulating the ubiquitination and degradation of viral nsP4

Alphavirus infection can result in a spectrum of clinical manifestations in the host, including fever, rash, arthritis, and even symptoms of encephalitis, thereby posing a severe threat to global public health security. In this study, we explored the role of arrestin domain-containing protein 1 (ARRDC1) in the replication of Semliki Forest virus (SFV), an important member of alphaviruses, by siRNA-based knockdown or CRISPR/Cas9-mediated knockout techniques. SFV replication levels are significantly increased by knockdown or knockout of ARRDC1 in multiple cell lines and inhibited by trans-complementation with ARRDC1. Our data further revealed that ARRDC1 affects the early RNA replication stage of SFV. The antiviral effect of ARRDC1 is dependent on its cell plasma membrane localization and ubiquitin ligase binding motif. Mechanistically, ARRDC1 binds to viral nonstructural protein 4 (nsP4) and facilitates its degradation by the ubiquitination pathway, thereby blocking the replication of SFV. In summary, this work identifies ARRDC1 as a novel restriction factor of SFV, potentially advancing the development of novel strategies against alphavirus infection.IMPORTANCESemliki Forest virus (SFV) belongs to the <i>Alphavirus</i> genus in the <i>Togaviridae</i> family and can cause a spectrum of clinical manifestations in the host, including fever, rash, arthritis, and even symptoms of encephalitis. Here, we reveal that ARRDC1 is a novel restriction factor for SFV in multiple cell lines, which relies on its cell plasma membrane localization and ubiquitin ligase binding motif. Interestingly, we further provide evidence that upon interacting with SFV nsP4, ARRDC1 mediates the degradation of nsP4 via the ubiquitination pathway, thereby inhibiting viral replication. Our study elucidates a new antiviral mechanism of ARRDC1 by mediating the ubiquitination and degradation of viral protein, which is of great significance for further understanding the pathogenesis of alphaviruses and the development of potential antiviral strategies.