Identification of YBX1 as an essential host RNA-binding protein governing tumor-selective replication of oncolytic...

Oncolytic virus M1 (OVM), currently in phase 1/2 clinical trials, is a single-stranded, positive-sense RNA virus. However, the host factors that engage its viral RNA to govern viral replication and oncolytic efficacy remain largely unknown. Here, by using RNA affinity purification coupled with mass spectrometry and CRISPR-Cas9 screening, we identify Y-box binding protein 1 (YBX1) as a predominant proviral RNA-binding protein. Functional dissection reveals that the cold shock domain of YBX1, specifically its aromatic residues W65, F74, and F85, are essential for binding OVM RNA and sustaining viral replication. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation sequencing maps that YBX1 binds to conserved sequence elements at the NSP4-capsid junction and 3' UTRs that function as promoters for viral RNA synthesis. Consequently, loss of YBX1 severely impairs viral RNA synthesis. This molecular dependency translates to a profound functional effect: across multiple tumor models, YBX1 deficiency abrogates OVM replication and oncolytic activity, while re-expression restores both. Importantly, YBX1 expression levels not only correlate with cellular susceptibility to OVM in cancer cell lines and ex vivo tumors but are also elevated in several human malignancies. Collectively, these findings establish YBX1 as a master regulator of OVM replication and a predictive biomarker for its therapeutic efficacy.