Degradation of miR-466d-3p by JEV NS3 facilitates viral replication and IL-1β expression

Abstract

27Previous studies revealed that Japanese encephalitis virus (JEV) infection alters the 28 expression of miRNA in central nervous system (CNS). However, the mechanism of JEV 29 infection contributes to the regulation of miRNAs in CNS remain obscure. Here, we found 30 that a global degradation of mature miRNA in mouse brain and neuroblastoma cells after JEV 31 infection. In additional, the integrative analysis of miRNAs and mRNAs suggests that those 32 down-regulated miRNAs are primarily targeted inflammation genes and the miR-466d-3p 33 target the IL-1β which in the middle of those inflammation genes. Transfection of 34 miR-466d-3p decreased the IL-1β expression and inhibited the JEV replication in NA cells. 35 Interestingly, the miR-466d-3p level increased after JEV infection in the presence of 36 cycloheximide, which indicated that viral protein expression reduces miR-466d-3p. 37Therefore, we generated all the JEV coding protein and demonstrated that NS3 is a potent 38 miRNA suppressor. Furthermore, the NS3 of ZIKA virus, WNV, DENV1 and DENV2 also 39 decreased the expression of miR-466d-3p. The in vitro unwinding assay demonstrated that the 40 NS3 could unwind the pre-miR-466d and induce the disfunction of miRNA. Using 41 computational models and RNA immunoprecipitation assay, we found that arginine-rich 42 domains of NS3 are critical for pre-miRNA binding and the degradation of host miRNAs. 43Importantly, site-directed mutagenesis of conserved residues revealed that R226G and 44 R202W significantly reduced the binding affinity and degradation of pre-miR-466d. 45 Together, these results extend the helicase of Flavivirus function beyond unwinding duplex 46 RNA to the decay of pre-miRNAs, which provides a new mechanism of NS3 in regulating 47 miRNA pathways and promoting the neuroinflammation. 48 49 Author Summary 50 Host miRNAs had been reported to regulate JEV induced inflammation in central nervous 51 system. We found that the NS3 of JEV can reduce most of host miRNA expression. The 52 helicase region of the NS3 specifically binds to precursors of miRNA and lead to incorrect 53 unwinding of precursors of miRNAs which inhibits the function of miRNAs. This observation 54 leads to two major findings. First, we identified the miR-466d-3p targets to the host IL-1β and 55 E protein of JEV, and NS3 degrades the miR-466d-3p to promote the brain inflammation and 56 viral replication. Second, we proved that the arginine on the helicase of NS3 is the main 57 miRNA binding sites, and the miRNA degradation by NS3 was abolished when the R226 and 58 R202 were mutated on the NS3. These findings were also confirmed with NS3 of ZIKA virus, 59WNV and DENV which could decrease the expression level of miR-466d-3p to enhance the 60 inflammation. Our study provides new insights into the molecular mechanism of encephalitis 61 caused by JEV, and reveals several amino acid sites to further attenuate the JEV vaccine. 62 63 65 JEV is a single-stranded, positive-sense RNA virus belonging to flavivirus of the Flaviviridae 66 family...