Host factor IRS4 impairs antitumor efficacy of oncolytic alphavirus via proteasomal viral protein degradation

Alphavirus M1, currently in phase I/II clinical trials (NCT07093814), provides a tractable system to define host determinants of oncolytic virus replication and efficacy. Tandem affinity purification-mass spectrometry mapped the nsP2 interactome in HEK293 cells and identified insulin receptor substrate 4 (IRS4) as a host restriction factor. Knockdown and reciprocal overexpression showed that IRS4 suppresses M1 replication and cytolysis without affecting baseline cell viability. Mechanistically, IRS4 engages the nsP23 replicase polyprotein in cells through an interaction interface mapped to the nsP2 C-domain and promotes ubiquitin-dependent proteasomal degradation of nsP23, thereby attenuating nsP3-dependent subgenomic RNA synthesis and viral structural protein production. TCGA profiling revealed heterogeneous IRS4 elevation in subsets of tumors, suggesting that endogenous IRS4 status may influence responses to M1 virotherapy. Across 12 human cancer cell lines, higher IRS4 expression showed a nonsignificant trend toward reduced M1 sensitivity, with higher logIC50 and AUC values. In PC9 and UMUC3 cells, ectopic IRS4 expression impaired M1-mediated killing in vitro and abrogated antitumor efficacy in xenograft models. Together, these findings identify IRS4 as a regulator of M1 replicase stability and support the hypothesis that IRS4 expression may represent a candidate negative biomarker for M1 oncolytic virotherapy.