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MiR-124 attenuates Japanese encephalitis virus replication by targeting DNM2.

ABSTRACT: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifier hosts of JEV. Emerging evidence indicates that host microRNAs (miRNAs) play key roles in modulating viral infection and pathogenesis. However, mechanistic studies delineating the roles of miRNAs in regulating host-JEV interactions remain scarce.In this study, we demonstrated that miR-124 inhibited JEV replication in porcine kidney epithelial PK15 cells. Furthermore, using bioinformatics tools, we identified dynamin2 (DNM2), a GTPase responsible for vesicle scission, as a target of miR-124. Small interfering RNA (siRNA) depletion studies inicated that dynamin2 was required for efficient JEV replication. We also demonstrated that upregulation of miR-124 expression corresponded to decreased expression of its target, DNM2, in the JEV-infected PK15 cells.Overall, these results suggest the importance of miR-124 in modulating JEV replication and provide a scientific basis for using cellular miRNAs in anti-JEV therapies.

SUBMITTER: Yang S

PROVIDER: S-EPMC4915174 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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miR-124 attenuates Japanese encephalitis virus replication by targeting DNM2.

Yang Songbai S Pei Yue Y Li Xinyun X Zhao Shuhong S Zhu Mengjin M Zhao Ayong A

Virology journal 20160621

<h4>Background</h4>Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifier hosts of JEV. Emerging evidence indicates that host microRNAs (miRNAs) play key roles in modulating viral infection and pathogenesis. However, mechanistic studies delineating the roles of miRNAs in regulating host-JEV interactions remain scarce.<h4>Results</h4>In this study, we demonstrated that miR-124 inhibited JEV replication in porci ...[more]

PMID: 27329300

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Project description:In flavivirus, the furin-mediated cleavage of prM is mandatory to produce infectious particles, and the immature particles containing uncleaved prM cannot undergo membrane fusion and release to the extracellular environment. However, the detailed relationship between viral replication or pathogenicity and furin in Japanese encephalitis virus (JEV) hasn't been clarified. Here, JEV with the mutations in furin cleavage sites and its nearby were constructed. Compared with WT virus, the mutant virus showed enhanced cleavage efficiency of prM protein and increased replication ability. Furthermore, we found that the mutations mainly promote genomic replication and assembly of JEV. However, the mutant formed smaller plaques than WT virus in plaque forming assay, indicating the lower cytopathogenicity of mutant virus. To assess the virulence of JEV mutant, an in vivo assay was performed using a mouse model. A higher survival rate and attenuated neuroinflammation were observed in JEV mutant-infected mice than those of WT-infected mice, suggesting the cleavage of prM by furin was closely related to viral virulence. These findings will provide new understanding on JEV pathogenesis and contribute to the development of novel JEV vaccines. IMPORTANCE Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis epidemics in Southeast Asia, affecting mostly children, with high morbidity and mortality. During the viral maturation process, prM is cleaved into M by the cellular endoprotease furin in the acidic secretory system. After cleavage of the prM protein, mature virions are exocytosed. Here, the mutant in furin cleavage sites and its nearby was constructed, and the results showed that the mutant virus with enhanced replication mainly occurred in the process of genomic replication and assembly. Meanwhile, the mutant showed an attenuated virulence than WT virus in vivo. Our study contributes to understanding the function of prM and M proteins and provides new clues for live vaccine designation for JEV.

Project description:UnlabelledJapanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. However, the molecular mechanism for JEV pathogenesis is still unclear. MicroRNAs (miRNAs) are small noncoding RNAs that act as gene regulators. They are directly or indirectly involved in many cellular functions owing to their ability to target mRNAs for degradation or translational repression. However, how cellular miRNAs are regulated and their functions during JEV infection are largely unknown. In the present study, we found that JEV infection downregulated the expression of endogenous cellular miR-33a-5p. Notably, artificially transfecting with miR-33a-5p mimics led to a significant decrease in viral replication, suggesting that miR-33a-5p acts as a negative regulator of JEV replication. A dual-luciferase reporter assay identified eukaryotic translation elongation factor 1A1 (EEF1A1) as one of the miR-33a-5p target genes. Our study further demonstrated that EEF1A1 can interact with the JEV proteins NS3 and NS5 in replicase complex. Through this interaction, EEF1A1 can stabilize the components of viral replicase complex and thus facilitates viral replication during JEV infection. Taken together, these results suggest that miR-33a-5p is downregulated during JEV infection, which contributes to viral replication by increasing the intracellular level of EEF1A1, an interaction partner of JEV NS3 and NS5. This study provides a better understanding of the molecular mechanisms of JEV pathogenesis.ImportanceMiRNAs are critical regulators of gene expression that utilize sequence complementarity to bind to and modulate the stability or translation efficiency of target mRNAs. Accumulating data suggest that miRNAs regulate a wide variety of molecular mechanisms in the host cells during viral infections. JEV, a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans worldwide. The roles of cellular miRNAs during JEV infections are widely unexplored. The present study explores a novel role of miR-33a-5p as a negative regulator of JEV replication. We found EEF1A1 as a direct target of miR-33a-5p. We also demonstrated that EEF1A1 interacts with and stabilize the components of JEV replicase complex, which positively regulates JEV replication. These findings suggest a new insight into the molecular mechanism of JEV pathogenesis and provide a possible therapeutic entry point for viral encephalitis.

Project description: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.

Dataset Information

MiR-124 attenuates Japanese encephalitis virus replication by targeting DNM2.

Publications

miR-124 attenuates Japanese encephalitis virus replication by targeting DNM2.

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