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

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


Introduction
replication, the turnover dynamics of miRNA and the mechanisms involved have been poorly 124 defined. murine cytomegalovirus (MCMV) directly degrade host miR-27 using a virus encode ncRNA repress the MCPIP1 to facilitate its own miRNA expression (29). Although the direct 136 interaction between host miRNAs and viral components remain far behind other studies, to 137 date, there are two approaches to predict RNA and protein interaction. One of the most 138 cost-effective approaches is computational predicting methods, which could predict 139 protein-RNA interaction and protein-RNA binding sites. Given the limited accurate of 140 computational methods, the RNA-protein immunoprecipitation provides a specific and 141 accurate predication of RNA-protein interaction. Based on those approaches, those would be 142 especially valuable to mapped miRNA-protein binding sites and characterized biological 143 function of viral components in viral replication process. 144 In the present study, JEV NS3 has been demonstrated to directly disrupt cellular pre-miRNA 145 and reduce mature miRNA levels. Through computational and RNA immunoprecipitation 146 (RIP) analysis, the miRNA binding and turnover of NS3 was found to be arginine-rich 147 domains dependent, which was determined by R226G and R202W of NS3. In addition, NS3 host miRNAs was altered during JEV infection (Fig. 1A). Notably, the 76.7% of total 41 158 miRNAs (p-value <0.05 and FC > 2.0) were reduced during JEV infection in mice brain. To 159 determine whether the global down regulation of endogenous miRNAs was biased in any 160 way, we used a small RNA deep sequence to analysis the miRNA expression profile of P3 161 infected NA cells at MOI of 1 and 0.1 for 48 hours (Fig. 1B). This analysis further confirmed 162 that JEV infection led to global host miRNAs decrease. Furthermore, the virus titer and NS3 163 expression level of cells infected with P3 at 1 MOI was higher than cell infected with P3 at 164 0.1 MOI, and 1 MOI infection more significantly decreased miRNA expression than 0.1 MOI 165 infection, suggesting that the JEV induced the global miRNA decrease is related to JEV 166 replication ( Fig. 1C and D). The miRNA profiles were also confirmed by qRT-PCR analyses 167 in JEV infected NA, BV2 and bEnd.3 cells (Fig. 1E). The integrative analysis of miRNAs and 168 mRNAs was used to predict the major target of the decreasing miRNAs. A total of 42 169 interacting proteins with 177 interactions were retrieved from the STRING database and 8 170 proteins were segregated to one subgroup, of which were related to immune and inflammation 171 process (S1 Fig). The result indicated that IL-1β is in the middle of these influential genes and 172 the miR-466d-3p is the only miRNA that decreased significantly (FC ≥ 2.0, p value < 0.01) 173 which target the IL-1β.
174 175 JEV infection induces incorrect processing of host miRNAs 176 Interestingly, the mature miRNA sequence-specific reads were determined by deep miR-466d-3p generated from miR-466d-3p precursor ( Fig. 2A), which were both significantly decrease the abundance of miR-466d-3p that derived from an exogenously 186 constructed miRNA mimic compared to the medium control in NA cells (Fig. 2C) were analysis with LC-MS (S1 Table). The amino sequence identification showed 209 234 proteins which score were higher than 90 and rich in several functional categories such as, IL-1β were significantly elevated in the dose-dependent and time-dependent manner in NA, 247 BV2 and bEnd.3 cells.1 (Fig. 4B).

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The results shown that overexpression of miR-466d-3p significantly suppressed IL-1β mRNA indicated that miR-466d-3p is a common regulator of IL-1β not specific to the JEV infection.

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Interestingly, a miRNA and JEV viral genomic gene comparison analysis indicated that the 262 miR-466d-3p also targets the coding sequence of JEV E, NS3 and NS5 genomes (Fig. 4C).

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There was a substantial reduction of virus titer in the NA cells that were transfected with 264 miR-466d-3p. Moreover, inhibition of miR-466d-3p leads to an increment of JEV replication 265 (Fig. 4G). To determine whether the miR-466d-3p direct target the seed sequence of JEV, we

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To further examine whether the unwinding miRNA could still transport into RISC and 288 execute a normal function of RNA silencing with host mRNA. The fragment of unwinding 289 miR-466d-3p mimic and pre-miR-466d-3p were recycled with Trizol after the in vitro 290 unwinding assay. After transfection with unwinding miR-466d-3p mimic, the expression level 291 of mature miR-466d-3p was similar to the NC control group, indicating that the unwinding 292 miR-466d-3p mimic was unstable in the host cells (Fig. 5D). Notably, we also did not observe 293 the mRNA expression and protein secretion level of IL-1β decreased after transfection of 294 unwinding miR-466d-3p mimic with or without JEV infection ( Fig. 5E and 5F). Taken 295 together, these data demonstrate that the NS3 of JEV could facilitate the disfunction of 296 miR-466d-3p in the host cells.
297 298 NS3 has specific binding affinity with pre-miRNA mediated by RNA-protein interactions (RPIs), and some computational models were degradation. In the present study, a RPISeq webserver was used to verify those hypotheses 305 (45). The Random Forest (RF) that calculated by RPISeq of indicated miRNAs between each 306 nonstructural protein of JEV was used to evaluate the RPI interaction. The probability 307 threshold of RF used for positive RPIs was higher than 0.5, and only the value of 308 pre-miRNAs or mature miRNAs that interacted with NS3 were all high than 0.5. The mean 309 RF of NS3 with pre-miRNA and mature miRNA was 0.675 and 0.69 respectively, and 310 significantly higher than all the other proteins of JEV (Fig. 6A).

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In contrast to the NS3, the miRNA binding ability of NS4A was similar to the pcDNA 3.1.

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However, in contrast to these in silico analysis, the RIP assay revealed that the binding ability 318 of pre-miR-466d-3p on NS3 was more than nonuple higher than miR-466d-3p and there is no 319 significant binding affinity of NS3 difference between the pcDNA3.1 and the NS4A ( were detected in NA cells with these three mutants or parent plasmids of NS3. As shown in 349 Fig. 7C, comparing with the pcDNA 3.1, the mutants of NS3 also specifically bind to 350 pre-miR-466d-3p, and the pre-miR-466d-3p binding ability of R226G and R202W were 351 significantly lower than wild type NS3 (Fig. 7C). Furthermore, the R226G and R202W 352 significantly reduce the degradation of miR-466d-3p, except for R464Q (Fig. 7D). Thus, the to explore the interplay between the JEV and host miRNAs.

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An intriguing aspect of this study is that the JEV globally decrease the host miRNA and 372 independent on Dicer or RISC. We demonstrated that the JEV NS3 could unwind the 373 pre-miR-466d and induce the disfunction of miR-466d-3p. We also observed that the  following the manufacturer's instruction using gene-specific primers (S2 Table).  Table). The pri-, pre-and mature-miRNA levels of cells were quantified 519 with the SYBR Green qPCR kit using miRNA sequence-specific primers (S4 Table and  gel, and protein blots were transferred to Nitrocellulose membrane (NC) after electrophoresis.

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The membrane was then washed with TBST and blocked in 5% skimmed milk at 4˚C 545 overnight. All primary antibodies were prepared at a dilution of 1:1,000 in 1% bovine serum 546 albumin (BSA) in 1×PBST, followed by horseradish peroxidase-conjugated secondary 2.5% trypsin and resuspended in 5ml PBS. 143μl of 37% formaldehyde was added to the 556 resuspension to cross-link for 10 minutes on a shaker, and 685μl of 2Mthe glycine was used 557 to block the formaldehyde. The cells were washed twice by 5ml ice-cold PBS and centrifuged and 5μl 40U/μl RNase inhibitor (Invitrogen) was added to cells, and the cells were kept on ice g and remove the supernatants. After the resin was washed three times with 0.5 ml 500μl of 566 TBS, the total RNA was extracted with Trizol reagent and analyzed by RT-qPCR.

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The fold change of each RIP reaction from RT-qPCR data was calculated via 2-△△CT 568 method as previously with minor modification (42) and the formulas see below. All the Ct MicroRNA target prediction 581 The sequences for the miRNA whose expression changed during JEV Infection have been 582 submitted to the public miRNA database miRBase (www.mirbase.org). The miR-466d-3p 583 target binding sites in the 3'UTRs of mouse gene transcripts were identified with Target Scan 584 software (http://www.targetscan.org/). The total RNA was isolated from mouse brains with Trizol reagent (Invitrogen) for miRNA 587 and mRNA Microarray. miRNA and mRNA hybridization were performed by shanghaiBio pair, the experiments were done with two independent hybridizations for miRNA (Agilent's (change fold ≥ 2.0, p value < 0.05) by JEV infection in mouse brain were clustered using GO 596 and KEGG enrichment tools (ShanghaiBio Analysis System). And the interaction of the most 597 significantly differential expression proteins was retrieved by STRING (59).  pre-miR-466d-3p processing and types of mature miRNAs (miR-466d-3p and miR-466h-3p).

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The blue arrow represented the miR-466h-3p located in the pre-miR-466d and the red arrow 911 represented the miR-466d-3p located in the pre-miR-466d. (B) Analysis of expression level of 912 mature miR-466d-3p, miR-466h-3p, pre-miR-466d-3p or pri-miR-466d using the quantitative has the opposite effect. The NA or BV2 cells was infected/treated with JEV at MOI of 0.01, of miR-466d-3p (VS negative control) by qRT-PCR. (E) After 6 hours transfection, the total