HAUS8 Regulates RLR-VISA Antiviral Signaling Positively By Targeting VISA

Innate immune system,developed in the long-term evolution process of organism,is essential for detecting the invading pathogens and the activation of subsequent specific immune,which plays an important function in the process of resist infection.Pathogen recognition receptors recruit adaptor proteins(PRRs)specificly by detecting conserved components of the pathogen or virus,causing a cascade of downstream signaling cascades,which induces antiviral respones and protects the host from infection.During a viral infection,the intracellular RIG-I-like receptors(RLRs)sense viral RNA and signal through VISA(also known as MAVS,IPS-1 and Cardif)whose activation triggers formation of the VISA complex on mitochondria,leading to a rapid production of type I interferons(IFNs)and of pro-inflammatory cytokines through activation of the transcription factors IRF3/IRF7 and NF-κB,respectively.VISA is the first protein found in mitochondria associated with innate immunity,which associated with peroxisomes,endoplasmic reticulum and autophagy.VISA plays an important role in antiviral or inflammatory pathways,and associated with apoptosis or metabolism.It remains to be elucidated how VISA work as a scaffold for protein complex assembly at the mitochondria to regulate the RLR-VISA antiviral signaling.Due to the important role in signal pathways,we identified 50 clones by using full length of VISA as bait to screen its interacting proteins in the 293 cell cDNA library by using yeast two-hybrid system.After analysis of sequencing and blast,expression frames and protein function prediction,We selected 4 candidate genes from 50 clones.We have cloned candidate genes to the mammalian cell expression vector(pRK5-Flag).We identified 3 candidate proteins(HAUS8(#13),#1 and #46)can interact with VISA by co-Immunoprecipitation,and HAUS8 was used to study the mechanism of antiviral using co-immunoprecipitation,double luciferase reporter gene system and RNA interference assay.In this study,we demonstrated that HAUS augmin like complex subunit 8(HAUS8)augments RLR-VISA dependent antiviral signaling pathway through targeting VISA complex.Co-immunoprecipitation(co-IP)verified that HAUS8 was associated with VISA and VISA signaling complex components RIG-I and TBK1 when RLR-VISA signaling pathway is activated.Our research data showed that overexpressiom of HAU8 significantly promotes the activities of transcription factor NF-кB,IRF3 and the IFN-β promoter induced by Sendai virus mediated RLR-VISA signaling.Knockdown of HAUS8 inhibits the activation of transcription factor IRF-3,NF-κB and the IFN-β promoter triggered by Sendai virus mediated RLR-VISA signaling.HAUS8 activates VISA complex by increased the polyubiquitination of VISA,RIG-I and TBK1.HAUS8 is a microtubule-associated protein important for maintaining spindle integrity and chromosomal stability.And,the study results demonstrated that HAUS8 functions as a positive regulator of RLR-VISA dependent antiviral signaling by targeting VISA complex for activation.HAUS8,as a novel cytoskeleton related protein involved in the RLR-VISA signaling pathway,provides a new regulating mechanism connecting cytoskeleton to RLR-VISA antiviral signaling.