Antiviral efficacy of short-hairpin RNAs and artificial microRNAs targeting foot-and-mouth disease virus

Abstract

RNA interference (RNAi) is a well-conserved mechanism in eukaryotic cells that directs post-transcriptional gene silencing through small RNA molecules. RNAi has been proposed as an alternative approach for rapid and specific control of viruses including foot-and-mouth disease virus (FMDV), the causative agent of a devastating animal disease with high economic impact. The aim of this work was to assess the antiviral activity of different small RNA shuttles targeting the FMDV RNA-dependent RNA polymerase coding sequence (3D). Three target sequences were predicted within 3D considering RNA accessibility as a major criterion. The silencing efficacy of short-hairpin RNAs (shRNAs) and artificial microRNAs (amiRNAs) targeting the selected sequences was confirmed in fluorescent reporter assays. Furthermore, BHK-21 cells transiently expressing shRNAs or amiRNAs proved 70 to >95% inhibition of FMDV growth. Interestingly, dual expression of amiRNAs did not improve FMDV silencing. Lastly, stable cell lines constitutively expressing amiRNAs were established and characterized in terms of antiviral activity against FMDV. As expected, viral replication in these cell lines was delayed. These results show that the target RNA-accessibility-guided approach for RNAi design rendered efficient amiRNAs that constrain FMDV replication. The application of amiRNAs to complement FMDV vaccination in specific epidemiological scenarios shall be explored further.

Keywords: Antivirals; FMDV; RNA interference; Target accessibility.

Figure 1. RNAxs results of target prediction within FMDV 3D region.

Data are represented as the number of predicted target sequences (coverage) per 3D nucleotide. Total number of predicted sequences (blue areas) and sequences corresponding to the top 50 hits in the RNAxs ranking (red areas) are shown. Target sequences selected for further study are indicated at the top of the corresponding peaks.

Figure 2. Silencing activity of small RNAs directed against FMDV 3D sequences.

Co-transfected cells were trypsinized and EGFP expression was analyzed by flow cytometry at 24 hpi as described in Material and Methods. Cells were co-transfected with pEGFP. 3D and shRNA_FMDV (A), pre-amiRNA_FMDV (B) or dual pre-amiRNA_FMDV (C) expressing plasmids. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. Antiviral effect of amiRNAs_FMDV stably expressed in BHK-21 cells.

(A) Cytopathic effect observed in BHK-21 cell lines expressing amiRNA_FMDV and control group (cell lines expressing miRneg) at 48 h post-infection. Cellular detachment, rounding, and destruction were more severe in the control group than in the experimental group (Magnification 100 ×). (B) Morphology and (C) dimensions of lysis plaques produced by FMDV in amiRNA_FMDV-expressing cell lines and control cells. FMDV infection produced less plaques of smaller dimensions in cell lines expressing amiRNA than in control cells. *** p < 0.0001. (D–F) Viral titers in supernatants of infected cells were determined by the TCID₅₀ method at 18 hpi (D), 24 hpi (E) or 48 hpi (F). ** p < 0.01.

Figure 4. Antiviral activity of cloned amiRNA₂₉₀ (A–C) and amiRNA₁₀₅₅ (D–F) cell lines.

Cell lines were obtained from polyclonal cell lines by limiting dilution as described in Material and Methods. Cells were infected with FMDV A/Arg/01 at a low moi and viral titers in supernatants at 18 hpi (A, D), 24 hpi (B, E) or 48 hpi (C, F) were determined by end-point dilution. * p < 0.05 (as compared to miRneg cells, Student’s t-test).