Zika Virus And Host Regulatory Network And MLK3 Participate In The Molecular Mechanism Of Virus Infection

Zika virus(ZIKV)is an emerging arthropod-borne virus in the genus Flavivirus and family Flaviviridae.It is a single-stranded RNA virus with an envelope protein.ZIKV was isolated in 1947 in the Zika Forest in Uganda from a sentinel Rhesus macaque.The virus activity has been very slightly.Since the first report in Brazil in early 2015,the ZIKV epidemic has spread rapidly in Central and South America,crossing the Pacific Ocean and reaching about 60 countries.Severe ZIKV infection can lead to neurological sequelae,such as microcephaly and Guillain-Barre syndrome,which has caused widespread concern.However,there are currently no vaccines or specific drugs against ZIKV.The ZIKV life cycle involves multiple steps including adsorption,penetration,uncoating,biosynthesis,assembly and release,and requires interactions with host factors.The interaction between virus and viral-associated host molecules lays the foundation for virus survival strategies and provides a powerful tool for identifying effective host targets and antiviral drugs.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 2659 small molecules and using the human glioblastoma cell line SNB-19 as an in vitro infection model,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 enhanced ZIKV infection in SNB-19 glioblastoma cells.Based on these different effects of the compound corresponding to the target protein,a network of interactions between ZIKV infection-associated regulatory factors was builed.Although a system-level network of ZIKV-associated proteins and cellular pathways was established,functional characterization is still needed.Among the hits,URMC-099,mixed lineage kinase 3(MLK3)inhibitor,significantly facilitated ZIKV replication in both SNB-19 cells and the neonatal mouse brain.MLK3 belongs to the serine/threonine kinase family.MLK3 phosphorylates and activates mitogen-activated protein kinase kinase(MAP2Ks).leading to JNK,P38,ERK(MAPKs)-mediated signaling pathway activation and participates in the regulation of cell proliferation,cell cycle progression and other processes.MLK3 can be activated by EV71 and hepatitis B virus.MLK3 deficiency delays influenza virus clearance in the lung.However,the role of MLK3 during ZIKV infection remains unknown.Using gene silencing and overexpression,we further confirmed that MLK3 was a host restriction factor against ZIKV.Mechanistically,MLK3 negatively regulated ZIKV replication through inducing the inflammatory cytokines IL-6,IL-8,TNF-a and MCP-1 but did not modulate IFN signaling pathway.Importantly,ZIKV replication in vitro and in the neonatal mouse brain activated the MLK3/MKK7/JNK pathway.Our work highlights the unique antiviral mechanism of MLK3 and suggests that protein agonists of the MLK3/MKK7/JNK signaling pathway may have clinical utility when re-used as ZIKV therapy.In summary,we obtained a high-throughput screening technique based on the function of a bioactive library with specific targets,systematically recognized ZIKV-infected host factors,and provided a theory for constructing a ZIKV-host interaction network to elucidate the regulatory mechanism of ZIKV infection.This study provides research directions for the discovery of ZIKV infection-associated biomarkers and new antiviral targets.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.