RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice

Abstract

RNA interference (RNAi) is a powerful tool to silence gene expression posttranscriptionally. In this study, we evaluated the antiviral potential of small interfering RNA (siRNA) targeting VP1 of foot-and-mouth disease virus (FMDV), which is essential during the life cycle of the virus and plays a key role in virus attachment to susceptible cells. We investigated in vivo the inhibitory effect of VP1-specific siRNAs on FMDV replication in BHK-21 cells and suckling mice, a commonly used small animal model. The results showed that transfection of siRNA-expressing plasmids gave an 80 to 90% reduction in the expression of FMDV VP1 in BHK-21 cells. Moreover, BHK-21 cells transiently transfected with siRNA-expressing plasmids were specifically resistant to FMDV infection when exposed to 100 50% tissue culture infective doses of virus, and the antiviral effects extended to almost 48 h postinfection. Furthermore, subcutaneous injection of siRNA-expressing plasmids in the neck made suckling mice significantly less susceptible to FMDV. In conclusion, our data suggests that RNAi may provide a viable therapeutic approach to treat FMDV infection.

FIG. 1.

Schematic representations of siRNA-expressing plasmids, target constructs, target viral mRNA, and predicted siRNAs. (A) An inverted repeat is inserted at the 3′ end of mouse the U6 promoter. The forward sequence of the repeat is 21 or 63 nt long, corresponding to the region of interest of the VP1 gene. The forward and reverse motifs are separated by a 6-nt spacer, 5′-CTCGAG-3′. The transcriptional termination signal of five Ts is added at the 3′ end of the inverted repeat. The synthesized RNA is predicted to fold back to form a hairpin dsRNA, which would be finally processed into the putative siRNAs. (B) To monitor the function of siRNAs, the cDNA of HKN/2002 VP1 was cloned into pEGFP-N1 and pcDNA3.1B(−) vectors as described in Materials and Methods. (C) The FMDV genome contains a unique open reading frame. The arrow at the bottom shows the site targeted by VP1-specific siRNAs.

FIG. 2.

Transient expression of siRNAs confers sequence-specific inhibition of expression of FMDV VP1 in BHK-21 cells. (A) Cells were cotransfected with pVP-EGFP-N1 and either pU6 or siRNA-expressing plasmids. As controls for nonspecific effects, cells were cotransfected with pEGFP and either pU6 or siRNA-expressing plasmids. Cells were visualized with a fluorescence microscope 24 h after transfection, and representative fields were photographed. (B) Inhibition of EGFP expression in cells cotransfected with pVP-EGFP-N1 (or pEGFP-N1) and siRNA-expressing plasmids. Cells were analyzed for EGFP expression by fluorescence-activated cell sorting, and the level of fluorescence relative to the control was quantitated. Data are the averages ± standard deviations from three separate experiments. (C) Cells were harvested, and transcripts were analyzed by RT-PCR amplification. The 28S, 18S, and 5.8S rRNAs were visualized under UV light for equal loading control.

FIG. 3.

Transient expression of siRNAs confers specific resistance against FMDV HKN/2002 in BHK-21 cells. Cells transfected with either pU6 or siRNA-expressing plasmids were infected with 100 TCID₅₀ of FMDV HKN/2002 per 0.1 ml. Cells were controlled by parallel assays with a divergent FMDV isolate (CHA/99) and an unrelated PRV isolate (Ea). After 24 h of infection, cells were visualized with an Olympus BH-2 microscope, and representative bright-field images were photographed.

FIG. 4.

BHK-21 cells transfected with pNT21 or pNT63 show a significant a reduction in virus yield, which is specific to HKN/2002 and extends to almost 48 h p.i. Cells transiently transfected with either pU6 or siRNA-expressing plasmids were infected with 100 TCID₅₀ of FMDV HKN/2002 (A), FMDV CHA/99 (B), or PRV Ea (C) per 0.1 ml, as described in Materials and Methods. Samples of supernatant were taken at different times p.i., and the virus titer (TCID₅₀) was determined three times on BHK-21 cells. Error bars indicate standard deviations.

FIG. 5.

siRNAs synthesized in vivo induce a rapid antiviral response in suckling mice and promote survival after FMDV infection. Suckling mice were challenged by subcutaneous injection in the neck with plasmid-virus complex containing 20 LD₅₀ of HKN/2002 (A) or 100 LD₅₀ of HKN/2002 (C). Alternatively, suckling mice were challenged by subcutaneous injection in the neck with 20 LD₅₀ of HKN/2002 (B) or 100 LD₅₀ of HKN/2002 (D) after 6 h of treatment with either pU6 or siRNA-expressing plasmids. All animals were observed for 5 days after challenge.

FIG. 6.

Preadministration of pVP1 promotes mouse survival after FMDV infection. Suckling mice were challenged by subcutaneous injection with 20 LD₅₀ of HKN/2002 after 6 h of treatment with 50 μg of pVP1 (or 50 μg of pNT21) or with a combination of pVP1 and pNT21 (or pVP1 and pNTH21) at the same dose and observed for 5 days.