Sui, Y.; Sherwood, M.; Okamoto, O. K.; Wang, Y.; Maringer, K.; Ewing, R. M.

Oncolytic virotherapy is an innovative approach to cancer treatment that uses replication competent viruses to selectively target and destroy cancer cells while leaving healthy tissues largely unaffected. Zika virus (ZIKV), a neurotropic orthoflavivirus, has recently gained attention as a potential oncolytic agent due to its ability to infect neural derived cells and suppress tumor growth in preclinical models. Although existing studies have examined ZIKV oncolytic effects, the mechanisms underlying these effects remain largely unexplored. Additionally, the roles of individual ZIKV proteins and their interactions with host factors remain incompletely understood. Here, we used RNA sequencing, affinity purification mass spectrometry, and functional assays to uncover previously unidentified mechanisms underlying ZIKV oncolytic activity in pediatric neural tumors. We found that the ZIKV non-structural proteins NS4A and NS5 exert oncolytic effects, reducing tumorsphere size. ZIKV host protein-protein interaction networks were characterized and showed that integrin 3 (gene: ITGA3), a mediator of cell matrix adhesion, interacts with ZIKV NS2B and NS4A. Integrin 3 was further shown to be involved in ZIKV and NS4A induced tumorsphere size reduction, while ITGA3 knockdown and ZIKV infection additively inhibited 3D invasion. These findings provide critical mechanistic insights that could inform the rational design of ZIKV based virotherapies and highlight opportunities for combination treatment strategies.