Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1β (IL-1β) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation. IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1β expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.
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...
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