Artificial MicroRNA-Mediated Inhibition of Japanese Encephalitis Virus Replication in Neuronal Cells

Abstract

Artificial microRNA (amiRNA)-mediated inhibition of viral replication has recently gained importance as a strategy for antiviral therapy. In this study, we evaluated the benefit of using the amiRNA vector against Japanese encephalitis virus (JEV). We designed three single amiRNA sequences against the consensus sequence of 3' untranslated region (3'UTR) of JEV and tested their efficacy against cell culture-grown JEV Vellore strain (P20778) in neuronal cells. The binding ability of three amiRNAs on 3'UTR region was tested in vitro in HEK293T cells using a JEV 3'UTR tagged with luciferase reporter vector. Transient transfection of amiRNAs was nontoxic to cells as evident from the MTT assay and caused minimal induction in interferon-stimulated gene expression. Furthermore, our result suggested that transient expression of two amiRNAs (amiRNA #1 and amiRNA #2) significantly reduced intracellular viral RNA and nonstructural 1 (NS1) protein, as well as diminished infectious viral particle release up to 95% in the culture supernatant as evident from viral plaque reduction assay. Overall, our results indicated that RNA interference based on amiRNAs targeting viral conserved regions at 3'UTR was a useful approach for improvements of nucleic acid inhibitors against JEV.

Keywords: 3′UTR; JEV; artificial microRNA; replication.

FIG. 1.

Cloning of amiRNA into pcDNA™6.2-GW/EmGFP-miR vector. (A) Schematic representations of the JEV 3′UTR structure. (B) Schematic illustrations of the three amiRNA designed against the target sites cloned into pcDNA6.2-GW/EmGFP-miR vector. amiRNA, artificial microRNA; JEV, Japanese encephalitis virus; UTR, untranslated region. Color images available online at www.liebertpub.com/nat

FIG. 2.

Transient transfection of amiRNAs and their effect on cell viability. (A) Cells seeded in a 96-well plate were infected with JEV at a MOI 5. Three hours postinfection, the cells were transfected with three different concentrations of amiRNAs (100, 500, and 1,000 ng) of single amiRNA per well. After 48 hpi, MTT reagent was added, and absorbance was measured at 570 nm. Results represent three independent experiments. (B) Cells were seeded in a 6-well plate and were transfected with four different concentrations of amiRNAs (50, 250, 500, and 1,000 ng) of single amiRNA per well. After 24 h, amiRNAs expression was monitored by checking eGFP expression under a fluorescence microscope. Representative images of amiRNA-treated HEK293T cells at 10 × magnification are shown. (C) RT-PCR analysis of four ISG (Ifitm1, Oasl1, Oasl2, and Isg15) mRNA expression. Gapdh gene expression was used to normalize the ISG expression. emGFP, emerald green fluorescent protein; hpi, hours postinfection; IFN, interferon; ISGs, IFN-stimulated genes; MOI, multiplicity of infection; qRT-PCR, quantitative real time polymerase chain reaction. Color images available online at www.liebertpub.com/nat

FIG. 3.

Binding of amiRNAs on 3′UTR of JEV Vellore strain (P20778) HEK293T cells were cotransfected with 100 ng JEV3′UTR luciferase construct and different concentration of amiRNA-expressing vector. After 48 h post-transfection, cells were lysed, and luciferase activity was measured. The data are shown as the firefly luciferase activity relative to the Renilla luciferase activity and are representative of three independent experiments. (A) Results represent the binding effect of 1,000 ng of amiRNA construct on JEV 3′UTR luciferase construct. The asterisk indicates statistical significance at 48 hpi (*P < 0.05). (B) Results represent the binding effect of different doses of amiRNA construct (50, 250, 500, and 1,000 ng) on JEV-3′UTR luciferase construct. (C) The nucleotide sequence of amiRNA #1, amiRNA #2, and amiRNA #5 (left panel) and their target site in JEV 3′UTR (right panel).

FIG. 4.

Effect of amiRNAs on JEV replication. Antiviral efficacy of each amiRNA was assessed in neuronal cells. (A) Determination of relative viral RNA by qRT-PCR. Cells were infected with cell culture-grown JEV (MOI = 5) and then treated with amiRNAs. After 48 hpi, cells were harvested, and RNA was isolated. The qRT-PCR was performed with specific primers for JEV region. Data represent the mean ± standard deviation of three independent experiments. (B) Western blots showing the amount of viral NS1 protein in neuronal cell lysate after treatment with different amiRNAs. Total cell lysate containing 30 μg proteins per sample was loaded. GAPDH served as an internal quantity and loading control. (C) Relative JEV titer in cell culture supernatant as measured by plaque assay at 48 hpi. (D) The 6-well plates are representing viral plaques after transfection of different amiRNAs. The number with a negative sign on each well represents 10 times dilution. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS1, Nonstructural 1; qRT-PCR, quantitative real time polymerase chain reaction. Color images available online at www.liebertpub.com/nat

FIG. 5.

Reduced viral NS1 expression in neuronal cells by amiRNAs. Representative images of amiRNA-treated N2a cells showed staining of JEV NS1 protein. The red fluorescence indicates the virus load as assessed with anti-JEV NS1 mAb and a secondary antibody conjugated with Alexa-594, and blue fluorescence suggests the nuclear staining with DAPI. The green fluorescence represents amiRNA expression into the cells. Color images available online at www.liebertpub.com/nat