Human MicroRNA hsa-miR-296-5p Suppresses Enterovirus 71 Replication by Targeting the Viral Genome

Abstract

Enterovirus 71 (EV71) has emerged as a major cause of neurological disease following the near eradication of poliovirus. Accumulating evidence suggests that mammalian microRNAs (miRNAs), a class of noncoding RNAs of 18 to 23 nucleotides (nt) with important regulatory roles in many cellular processes, participate in host antiviral defenses. However, the roles of miRNAs in EV71 infection and pathogenesis are still unclear. Here, hsa-miR-296-5p expression was significantly increased in EV71-infected human cells. As determined by virus titration, quantitative real-time PCR (qRT-PCR), and Western blotting, overexpression of hsa-miR-296-5p inhibited, while inhibition of endogenous hsa-miR-296-5p facilitated, EV71 infection. Additionally, two potential hsa-miR-296-5p targets (nt 2115 to 2135 and nt 2896 to 2920) located in the EV71 genome (strain BrCr) were bioinformatically predicted and validated by luciferase reporter assays and Western blotting. Genomic alignment of various EV71 strains revealed synonymous mutations in hsa-miR-296-5p target sequences. Furthermore, the introduction of synonymous mutations into the EV71 BrCr genome by site-directed mutagenesis impaired the viral inhibitory effects of hsa-miR-296-5p and facilitated mutant virus infection. Meanwhile, compensatory mutations in corresponding hsa-miR-296-5p target sequences of the EV71 HeN strain (GenBank accession number JN256064) restored the inhibitory effects of the miRNA. These results indicate that hsa-miR-296-5p inhibits EV71 replication by targeting the viral genome. Our findings support the notion that cellular miRNAs can inhibit virus infection and that the virus mutates to escape suppression by cellular miRNAs.

Transcriptional Suppression of miR-181c by Hepatitis C Virus Enhances Homeobox A1 Expression

“…On the other hand, miR-196, miR-351, miR-431, and miR-448 inhibit HCV replication in Huh7 cells by interacting with the HCV genome (36). Recently, miR-296-5p was shown to inhibit enterovirus 71 replication by binding to the VP-1 gene (37). Our study demonstrated that the introduction of miR-181c into HCVinfected hepatocytes or cells harboring the HCV genome-length replicon inhibits viral genome replication.…”

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

“…Recombinant EV71 mutants harboring 3D mutations were constructed by overlapping extension PCR using the infectious clone as a template at the indicated sites. Verification of the mutants was performed by RNA extraction, reverse transcription, and PCR (39).…”

“…The EV71 infectious clone and its mutant recombinant RNAs were in vitro transcribed with a RiboMax large-scale RNA production system T7 kit (Promega) (43). The RNAs were transfected into Vero cells to rescue EV71 wild-type (WT) and mutant viruses, and these viral strains were amplified in RD cells (39).…”

“…Our findings together imply that EV71 exploits host cellular modification for effective replication, revealing a potential target for antiviral therapy. (39). Virus titers were measured by 50% tissue culture infectious dose (TCID 50 ) in RD cells using the ReedMuench formula (40).…”

Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein

“…miR-122, a liver-specific miRNA, interacts with the 5= UTR of the genome of the hepatitis C virus (HCV) and increases viral replication (13). miR-296-5p and miR-141 are upregulated to modulate EV71 replication by targeting the viral genome and host eIF4E, respectively (14,15).…”