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.

Fig 1

Expression of hsa-miR-296-5p and hsa-miR-196 in EV71-infected human RD and SK-N-SH cells. (A, B) At different times after EV71 infection, levels of hsa-miR-296-5p and miR-196 were quantified by qRT-PCR, using U6 rRNA as an internal control. Values are means from triplicate experiments and represent relative levels of miRNA in RD (A) and SK-N-SH (B) cells. (C, D) To determine expression of miRNA on a per cell basis, 15 pg of total RNA extracted from EV71-infected cells at each time point was used to determine the copy numbers of miR-296-5p and miR-196 in RD (C) and SK-N-SH (D) cells. Chemically synthesized oligoribonucleotides corresponding to miR-196b and miR-296-5p were used to generate standard curves. (E) IFN receptors of EV71-infected SK-N-SH cells were blocked by mouse anti-IFNAR monoclonal antibody. Relative expression levels of hsa-miR-296-5p were determined by qRT-PCR at different times. The untreated group (EV71) and IgG-treated group (IgG+EV71) were used as a control. Data are representative of at least two independent experiments, with each determination performed in triplicate (mean ± SD of fold change). *, P < 0.05; **, P < 0.01.

Fig 2

Effects of hsa-miR-296-5p overexpression on EV71 infection. RD cells were transfected with synthetic hsa-miR-296-5p mimics or the negative-control mimics (NC). (A) Titration of EV71 in cells transfected with hsa-miR-296-5p or NC. Virus titers in infected cells were determined at the indicated times postinfection. (B, D) EV71 RNA expression in hsa-miR-296-5p-transfected cells during viral infection. After extraction of total RNA from infected cells in hsa-miR-296-5p- and NC-transfected groups, EV71 genomic RNA levels were determined by qRT-PCR and normalized against GAPDH transcript levels (B), and levels of hsa-miR-296-5p were quantified by qRT-PCR, using U6 rRNA as an internal control (D). (E) SK-N-SH cells transfected with miR-296-5p mimics and negative control were subjected to Ago2-IP, and copy numbers of miR-296-5p and miR-196 in the immunoprecipitates were determined by qRT-PCR. As a negative control, immunoprecipitation was performed using nonimmune IgG beads. The symbols “+” and “−” indicate treatment and no treatment, respectively, by the corresponding factor. Data are representative of at least two independent experiments, with each determination performed in triplicate (mean ± SD of fold change). *, P < 0.05; **, P < 0.01. (C) Viral protein expression in hsa-miR-296-5p-transfected cells during EV71 infection. Proteins of treated cells were extracted at the indicated times postinfection. Equal amounts of cell lysates were analyzed by immunoblotting with an anti-EV71-VP1 (upper panel) or anti-β-actin (lower panel) antibody. Results shown are representative of three independent experiments.

Fig 3

Effects of hsa-miR-296-5p-mediated inhibition of EV71 infection. SK-N-SH cells were transfected with a synthetic hsa-miR-296-5p inhibitor (inhibitor) or the NC inhibitor. (A) Titration of EV71 in infected cells transfected with hsa-miR-296-5p or NC. Virus titers in infected cells were determined at the indicated times postinfection. (B) EV71 RNA levels in hsa-miR-296-5p-transfected cells during viral infection. After extraction of total RNA from infected cells in inhibitor- and NC-transfected groups, EV71 genomic RNA levels were determined by qRT-PCR and normalized against GAPDH transcript levels. Data are representative of at least two independent experiments, with each determination performed in triplicate (mean ± SD of fold change). *, P < 0.05; **, P < 0.01. (C) Viral protein expression in hsa-miR-296-5p-transfected cells during EV71 infection. Proteins of treated cells were extracted at the indicated times postinfection. Equal amounts of cell lysates were analyzed by immunoblotting with an anti-EV71-VP1 (upper panel) or anti-β-actin (lower panel) antibody. Results shown are representative of three independent experiments.

Fig 4

Targeting of EV71 genomic RNA sequence by hsa-miR-296-5p. (A, B) The complementary sequences of two hsa-miR-296-5p candidate target “seed sequences” and mutated forms are indicated. Mutated nucleotides are bolded. The complementary sequences are from the EV71-BrCr-TR strain (GenBank number AB204852). (C, D) Luciferase reporter plasmids containing the hsa-miR-296-5p target sites (2115-luc and 2896-luc) and corresponding mutants (m2115-luc and m2896-luc) were cotransfected with the plasmid pRL-TK into hsa-miR-296-5p- or NC-treated RD cells. Reporter activities were determined 24 h posttransfection by dual-luciferase assays, and the resultant ratios are shown. Data are representative of at least two independent experiments, with each determination performed in triplicate (mean ± SD of fold change). *, P < 0.05; **, P < 0.01. (E, F) Schematic representation of constructs Flag-VP1 and Flag-VP3 and corresponding constructs Flag-VP1m and Flag-VP3m containing mutations in hsa-miR-296-5p target sites. (G, H) Western blot analysis of effects of hsa-miR-296-5p on expression of EV71 VP1 and its mutant VP1m (G) and VP3 and its mutant VP3m (H). RD cells were transfected with the indicated plasmids in the presence of hsa-miR-296-5p or randomized NC. Proteins were analyzed with an anti-FLAG antibody with β-actin as the internal control.

Fig 5

Effects of synonymous mutations in hsa-miR-296-5p target sites on EV71 replication and inhibitory effects of hsa-miR-296-5p on EV71. (A) Genomic sequence alignment of EV71 strains of different subtypes. Synonymous nucleotide mutations in the seed region of the two hsa-miR-296-5p target sites are boxed. (B) Schematic representation of pEV71(BrCr-TR) constructs with the hsa-miR-296-5p target sites and their corresponding mutants. (C) Sequencing results of each mutant EV71 virus at mutation sites. (E) Effects of hsa-miR-296-5p on infectivity of WT (BrCr) and mutant (mt2115, mt2896, and double mutant) viruses. hsa-miR-296-5p-transfected RD cells were infected with each virus at an MOI of 5. Viral titers were determined at 12 h postinfection. Data are representative of at least two independent experiments, with each determination performed in triplicate (mean ± SD of fold change). **, P < 0.01. (D) Growth curves of WT and mutant viruses. SK-N-SH cells were infected with each virus at an MOI of 5, and EV71 titers of infected cells were determined. Data are representative of at least two independent experiments, with each determination performed in triplicate (mean ± SD of fold change). **, P < 0.01.

Fig 6

Effects of back mutations in hsa-miR-296-5p target sites on replication and inhibitory effects of hsa-miR-296-5p on EV71-HeN-luc. (A) Schematic representation of pEV71-HeN-luc constructs with hsa-miR-296-5p target sites and their corresponding back mutations. (B) Sequencing results of each mutant EV71 virus at the two mutation sites. (C) Effects of hsa-miR-296-5p on infectivity of WT (HeN) and mutant (HeN-2115, HeN-2896, and HeN-MD) viruses. hsa-miR-296-5p-transfected RD cells were infected with each virus at an MOI of 5. Viral titers were determined at 12 h postinfection. Data are representative of at least two independent experiments, with each determination performed in triplicate (mean ± SD of fold change). **, P < 0.01.