The Regulation And Mechanism Of MiR-142a-5p In The Neurological Injury Induced By Porcine Hemagglutinating Encephalomyelitis Virus

Porcine hemagglutinating encephalomyelitis,an acute and highly contagious disease in pigs,is caused by porcine hemagglutinating encephalomyelitis virus(PHEV).PHEV is a neurotropic coronavirus that invades the central nervous system(CNS)and causes neurological dysfunction.The discovery of microRNAs(miRNAs)has greatly expanded our understanding of the neuropathogenesis of PHEV.Both DNA and RNA viruses have evolved mechanisms to degrade,boost,or hijack cellular mi RNAs to benefit the viral life cycle or fine-tuning the host homeostasis.Previous work on miRNA/mRNA interaction networks in PHEV-infected mice have uncovered that host miRNAs(miR-142a-5p,miR-10a-5p,miR-763,etc.)significantly altered during neurological dysfunction.Therefore,we have explored the functions and regulation of miR-142a-5p,providing a better understanding of PHEV pathogenesis at a transcriptomic level.Here,we established models of PHEV-infected mice and primary cortical neuron,providing an ideal support in vivo and vitro system.Using the above infection models,we then confirmed that PHEV significantly up-regulated mi R-142a-5p level,suggesting PHEV largely exploited spatiotemporal control of host miRNAs for neuropathogenicity.Identification of target genes is the basis for the study of miRNA functions,thus,we sought to screen mi R-142a-5p target mRNAs.We have predicted 106 potential targets by using bioinformatics databases,including TargetScanMouse 6.2,mi Randa,MicroT-CDS,and PicTar.Valued these mRNAs by GO,KEGG,and Cytoscape analysis,we found that Unc-51-like kinase 1(Ulk1)was of particular interest and contained conserved 3'UTR sequence elements targeted by miR-142a-5p.We first demonstrated the loss of Ulk1 expression during PHEV infection using by qRT-PCR,CLSM,and WB analyses.CLSM analysis also showed a co-localization expression of miR-142a-5p and Ulk1 in the neuronal body and neurites,implying of an interrelationship.Modulating miR-142a-5p activity by mimics/inhibitors showed that miR-142a-5p negatively regulated Ulk1 expression in a dose-dependent manner.Using EMSA and DLR activity analysis further confirmed that miR-142a-5p specifically targeting to Ulk1 mRNA 3'UTR region,which is one of the downstream targets for mi R-142a-5p that involved in regulating PHEV-associated neurological dysfunction microenvironment.To value the important role of miR-142a-5p-mediated Ulk1 mRNA repression in PHEV infection,we investigated the life cycle of PHEV.Study on miR-142a-5p inhibitors transfection revealed that inhibition of mi R-142a-5p promoted the replication and proliferation of PHEV,and injection of mi R-142a-5p blockmirs(antagomir)into the cerebral cortex of PHEV-infected mice showed a certain protective effect.Using TEM and DiD-labeling tracer analysis proved that PHEV was internalized into the Rab5 early endosomes(EEs)and hijacked the host endosomal transport system for intracellular traffic.To gain insight into the possible mechanisms,we tested whether Ulk1 dysfunction affected Rab5-mediated PHEV internalization and intracellular transport.Treatments of PHEV infection,mi R-142a-5p mimics/inhibitors transfection,and Ulk1 RNAi have proved that Ulk1 deficiency increases the activity of Rab5 GTPase and promotes PHEV internalization and proliferation.We then constructed the wild-type EGFP-Rab5 wt,dominant active mutant EGFP-Rab5S34 N,and dominant negative mutant EGFP-Rab5Q79 L,and transfected them into neurons,respectively.The results proved that PHEV infection depends on the regulation of Rab5 GTPase activity by Ulk1.Since miR-142a-5p pivotal functions in PHEV infection,we next tested whether it was already involved in PHEV-induced abnormal neuron morphology.Therefore,we applied the CRISPR/Cas9 system to modify the Ulk1 gene at the DNA level,and overexpressed replication-deficient adenoviruses encoding Ulk1(Ad5-GFP-Ulk1)in the cortical neurons.Studies on these systems have shown that Ulk1 plays a vital role in promoting neuronal growth and development,but the Ulk1 dysfunction induced neurodegeneration,which characterized by stunted axon extension,excessive branch,neuritic beading,and disconnected neuritis,consistenting with the expected defects that occur during PHEV infection.To further reveal the neuropathologic mechanism of Ulk1-deficient,we then explored the role of Ulk1 and its downstream factor Rab5 in signal transduction of nerve growth factor(NGF).Stimulation with Cy3-NGF suggested that PHEV significantly inhibited the phosphorylation of NGF high affinity receptor TrkA,and blocked Ulk1-mediated endocytic of NGF.Rescued by high concentration of NGF,it was proved that Ulk1-mediated NGF/TrkA internalization controls the process of PHEV-induced neuronal morphological abnormalities.Co-IP and CLSM analysis in Rab5 mutation neurons proved that lacking of Ulk1 function promotes NGF/TrkA to be internalized into activated Rab5 GTP EEs and contributes to axonal transport defects,thereby leading NGF/TrkA signal transduction disorder.Given that NGF is pivotal for neuronal outgrowth and differentiation,we suggested that Ulk1-mediated NGF/TrkA signaling pathway disorder is one of the important mechanisms of PHEV-induced neurological dysfunction.Together,our findings reveal mi R-142a-5p-mediated Ulk1 repression functions in PHEV-induced neurodegenerative disease.On the one hand,Ulk1 dysfunction promotes Rab5 GTPase activating,which allows PHEV internalization and intracellular transport in Rab5-dependent endocytic pathways.On the other hand,Ulk1 repression results in limiting NGF/TrkA retrograde signal transduction within activated Rab5 GTP EEs,explaining the progressive failure of neurite outgrowth and survival.Our data not only contribute greatly to neuropathogenicity of PHEV,but also enhance our understanding of how the neurotropic viruses disturb the CNS through post-transcriptional events.