MicroRNA-134 regulates poliovirus replication by IRES targeting

Figures

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  Figure 1. Sabin-1 replication is inhibited by miR-134 post-infection. (a) HEp-2 cells were infected with Sabin-1, Sabin-2, or Sabin-3 and transfected with control or a miR-134 mimic (25 nM). Cell supernatants were assayed for viral titers on HEp-2 cells. Error bars represent ± SEM from two independent experiments with triplicate replicates each. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001. Viral titers are shown as log 10 pfu/ml. (b) Data in (a) are shown on a linear scale.

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  Figure 2. MiR-134 interacts with Sabin-1 and Sabin-3 but not Sabin-2. Absorbance profiles of renatured Sabin-1, Sabin-2, Sabin-3 oligonucleotides alone or with miR-134 were measured using UV spectrophotometry. Profiles represent hypochromic shifts in case of Sabin-1 (a) and Sabin-3 (c) and a hyperchromic shift for Sabin-2 (b). Data represent means of multiple measurements on six independent assays. Alignments show base pairing between viral regions and miR-134. Solid lines indicate Watson-Crick base pairs while colon symbols represent non-Watson-Crick pairs.

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  Figure 3. Reporter assays validate miR-134 inhibition of Sabin-1 but not Sabin-2 or Sabin-3 translation. (a) Design of reporter constructs for Sabin-1, Sabin-2, and Sabin-3 plasmids. Binding site for miR-134 is indicated for Sabin-1 in red (loop V). (b) Alignments of miR-134 and Sabin-1, Sabin-2 and Sabin-3 IRES are shown. Solid lines indicate Watson-Crick base pairs while colon symbols represent non-Watson-Crick pairs. Bold residues indicate miR-134 seed site. (c) Constructs above were co-transfected with control/miR-134 mimic and pSEAP2 Control (transfection control plasmid) into HEp-2 cells using Lipofectamine 2000. Luciferase expression was determined 48 h post transfection and normalized to SEAP expression. Control mimic (MNC1) values represent 100%. Error bars represent ± SEM from two independent experiments with triplicate replicates each. *p < 0.05, ****p < 0.001.

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  Figure 4. Site directed mutagenesis validates miR-134 targeting of the Sabin-1 IRES. (a) Alignments of wild type and mutated Sabin-1 and Sabin-2 sequences are shown aligned to miR-134 sequence. Solid lines indicate Watson-Crick base pairs while colon symbols represent non-Watson-Crick pairs. Bold residues in Sabin-1/2 sequences indicate residues that were mutated. (b) Sequence verified endotoxin free wild type (wt)/mutant (mut) plasmids were co-transfected into HEp-2 cells with control/miR-134 mimic and pSEAP2C control plasmid. Luciferase expression was assayed at 48 h post transfection. Mimic control (MNC1) values are set to 100%. Error bars represent ± SEM from two independent experiments in quadruplicate. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001. ns = non-significant.

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  Figure 5. MiR-134 binding causes degradation of Sabin-1 IRES. HEp2c cells were co-transfected with endotoxin-free Sabin-1 wild type (S1wt) or mutant (S1 mut) IRES-luciferase and miR-134 mimic (134 M)/MNC1 (25 nM) for 24 h. Stability of Sabin-1 IRES was determined by Sabin-1 IRES specific PCR from equal amounts of cDNA from DNAse I treated total RNA. PCR products were resolved on 1% agarose gels containing 0.5 µg/ml Ethidium bromide in 1X TAE buffer at 3 V/cm. Image shows region of gel containing band of expected size representative of two independent experiments. Marker size is in kb.

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  Figure 6. Sequence logos of miR-134 binding sites in PV-1and EV71 but not PV-2 IRES regions show extensive conservation. Genomes of wild type Sabin-1 (n = 218), Sabin-2 (n = 284) and EV71 (n = 615) sequences were aligned using CLUSTALW and conservation if any of putative miR-134 binding site was analyzed using WebLogo 3.0. The sequences represent consensus miR-134 binding site as predicted by computational algorithms in these regions of the PV-1, PV-2 and EV71. The height of each letter corresponds to degree of conservation in the dataset at the position examined.