High-Throughput Screening Identifies Mixed-Lineage Kinase 3 as a Key Host Regulatory Factor in Zika Virus Infection

Abstract

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-Barré 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.

Pyroptosis and ferroptosis induced by mixed lineage kinase 3 (MLK3) signaling in cardiomyocytes are essential for myocardial fibrosis in response to pressure overload

“…As a mixed lineage kinase inhibitor, URMC-099 possesses the ability to inhibit the activity of MLK1, MLK2, LRRK2, and MLK3 34 , 48 , which could act as a useful tool to investigate the role of MLK3 in many other diseases 38 – 40 . Interestingly, the similar effects of URMC-099 on an established cardiac remodeling were observed in the present study.…”

“…C57BL/6J male mice were randomly assigned to eight groups: Sham, TAC, TAC + U-099, Sham + AAV NC , TAC + AAV NC , TAC + AAV MLK3− , TAC + atagomir, and TAC + agomir. TAC + U-099 mice were generated by intraperitoneal (i. p.) injection of URMC-099, an inhibitor of MLK3, has been reported to play an important role in a variety of diseases including anti-inflammatory and cognitive decline 34,37 and as a useful tool to investigate the role of MLK3 in other diseases is common [38][39][40] (10 mg/kg, dissolved in 10% DMSO, 40% PEG300, and 50% saline, every 12 h, MedChemExpress, Shanghai, China) 7 days before TAC surgery. Sham and TAC mice received corresponding isotype i.p.…”

Zika Virus Pathogenesis: A Battle for Immune Evasion

“…Many of these functional sites have evolved to act against similar factors and functions making the host immune system antagonism redundant but adequately efficient, due to the need to competently escape the immune surveillance and to establish infection in the host [ 173 , 174 ]. Variability at these sites may modulate ZIKV infection capacity, optimization of immune evasion and pathogenicity, and this could be associated with the infection severity degree of the different viral phenotypes or lineages [ 140 , 142 , 175 , 176 , 177 , 178 ].…”

ANKS4B Restricts Replication of Zika Virus by Downregulating the Autophagy

“…Role of many proteins in ZIKV replication have been elucidated, such as α2,3-linked sialic acid which facilitates virus internalization ( Tan et al, 2019 ), heat shock protein 70 ( Pujhari et al, 2019 ), endoplasmic reticulum (ER) membrane protein complex ( Barrows et al, 2019 ), adenosine deaminases acting on dsRNA 1 ( Zhou et al, 2019 ), stearoyl-CoA desaturase-1 ( Hishiki et al, 2019 ), and fibroblast growth factor 2 ( Limonta et al, 2019 ). On the other hand, host cell elicits a variety of responses against ZIKV, including innate immune response, cell death, unfolded protein response, and stress granule formation, during which a number of cellular factors particularly the IFN-stimulated genes (ISG) are involved in, such as cholesterol-25-hydroxylase ( Doms et al, 2018 ), PARP12 ( Li et al, 2018 ), mixed-Lineage Kinase 3 ( Xu et al, 2019 ; Yang et al, 2019 ), E3 ligase TRIM56 ( Yang et al, 2019 ), and schlafen 11 ( Valdez et al, 2019 ).…”