High-throughput screening identifies mixed lineage kinase 3 as a key host regulatory factor in Zika virus infection

: The Zika virus (ZIKV) life cycle involves multiple steps and requires interactions with host factors. However, the inability to systematically identify host regulatory factors for ZIKV has hampered antiviral development and our understanding of pathogenicity. Here, using a bioactive compound library with 2,659 small molecules, we applied a high-throughput and imaging-based screen to identify host factors that modulate ZIKV infection. The screen yielded hundreds of hits that markedly inhibited or potentiated ZIKV infection in SNB-19 glioblastoma cells. Among the hits, URMC-099, a mixed-lineage kinase 3 (MLK3) inhibitor, significantly facilitated ZIKV replication in both SNB-19 cells and the neonatal mouse brain. Using gene silencing and overexpression, we further confirmed that MLK3 was a host restriction factor against ZIKV. Mechanistically, MLK3 negatively regulated ZIKV replication through induction of the inflammatory cytokines interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein 1 (MCP-1) but did not modulate host interferon-related pathways. Importantly, ZIKV activated the MLK3/MKK7/Jun N-terminal protein kinase (JNK) pathway in both SNB-19 cells and neonatal mouse brain. Together, these findings reveal a critical role for MLK3 in regulating ZIKV infection and facilitate the development of anti-ZIKV therapeutics by providing a number of screening hits.IMPORTANCE Zika fever, an infectious disease caused by the Zika virus (ZIKV), normally results in mild symptoms. Severe infection can cause Guillain-Barre syndrome in adults and birth defects, including microcephaly, in newborns. Although ZIKV was first identified in Uganda in 1947 in rhesus monkeys, a widespread epidemic of ZIKV infection in South and Central America in 2015 and 2016 raised major concerns. To date, there is no vaccine or specific medicine for ZIKV. The significance of our research is the systematic discovery of small molecule candidates that modulate ZIKV infection, which will allow the development of antiviral therapeutics. In addition, we identified MLK3, a key mediator of host signaling pathways that can be activated during ZIKV infection and limits virus replication by inducing multiple inflammatory cytokines. These findings broaden our understanding of ZIKV pathogenesis.