Silencing of the foot-and-mouth disease virus internal ribosomal entry site by targeting relatively conserved region among serotypes
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Foot-and-mouth disease (FMD) is a host-restricted disease of cloven-hoofed animals, such as cattle and pigs. There are seven major serotypes of FMD virus that exhibit high antigenic variation, making vaccine strain selection difficult. However, there is an internal ribosomal entry site (IRES) element within the 5′ untranslated region of the FMD virus (FMDV) RNA genome that is relatively conserved among FMDV serotypes and could be used as a pan-serotype target for disease interventions. To determine the potential for targeting the IRES as promising drug target, we designed a short interfering RNA (siRNA) targeting a relatively conserved region in the FMDV-IRES. The siRNA affected FMDV-IRES expression but not the expression of the encephalomyocarditis virus or hepatitis C virus IRES. To evaluate the effects of siRNA-mediated silencing, we established cell lines expressing a bicistronic luciferase reporter plasmid, which contained an FMDV-IRES element between the Renilla and firefly luciferase genes. The designed siRNA inhibited FMDV-IRES-mediated translation in a concentration-dependent manner. In order to sustain this inhibitory effect, we designed a short hairpin RNA (shRNA)-expressing lentiviral vector. The results showed that the lenti-shRNA vector significantly suppressed FMDV-IRES activity for up to 2 weeks in cell culture. Thus, our findings in this study provided a basis for the development of effective pan-serotype FMDV inhibitors.
KeywordsFoot-and-mouth disease virus Internal ribosomal entry site Short interfering RNA Short hairpin RNA Translation
Foot-and-mouth disease (FMD) virus (FMDV; genus Aphthovirus, family Picornaviridae) is a positive-sense, single-stranded RNA virus that causes FMD, a highly contagious disease of cloven-hoofed animals. FMD is epidemic or sporadic in numerous countries , and seven serotypes of FMDV, i.e., O, A, C, Asia 1, SAT1, SAT2, and SAT3 , have been identified. FMDV isolates show high levels of genetic diversity . Serotype O is most prevalent, followed by serotype A . The multiple serotypes and variants make disease control difficult; indeed, antigenic differences within a serotype are so great that little or no cross-protection can be achieved between strains of the same serotype . Consistent with these observations, some capsid proteins exhibit sequence variation, with VP1 varying by approximately 30–50% among serotypes .
There is an internal ribosomal entry site (IRES) element within the 5′ untranslated region (5′UTR) of the FMDV RNA genome, and this IRES mediates the translation of viral proteins [7, 8]. Other picornaviruses, such as poliovirus (PV) and encephalomyocarditis virus (EMCV), and flaviviruses, such as hepatitis C virus (HCV), possess virus-specific IRES elements within their 5′UTRs [9, 10]. IRES can be classified into five types, designated I (PV), II (FMDV), III (hepatitis A virus), IV (HCV-like), and V (aichivirus-like), based on the higher-order structure . Most eukaryotic mRNAs are translated in a cap-dependent manner, which involves recognition of the 5′ cap structure by the 43S ribosome . Viral mRNA has a short 5′UTR (< 100 nucleotides) and does not contain an initiation AUG, enabling protein synthesis in a cap-dependent manner, similar to most eukaryotic mRNAs [9, 10]. In contrast, IRES-mediated translation is cap-independent [9, 10]. The translation of eukaryotic mRNA is halted by the cleavage of eIF4G with picornavirus protease (e.g., PV 2Apro and FMDV Lpro), whereas protein synthesis regulated by PV or EMCV-IRES is stimulated [13, 14]. FMDV Lpro can enhance translation targeting all picornavirus IRES, even after the inactivation of eIF2 by phosphorylation . Because the IRES region is responsible for translational control functions, its nucleotide sequence is relatively conserved among FMDV serotypes.
In this study, we developed an approach to silence FMDV-IRES by targeting a relatively conserved region among the seven FMDV serotypes in order to gain basic information for the establishment of pan-serotype inhibitors.
Materials and methods
Cell culture and plasmids
The human kidney cell line (HEK293) used in this study was obtained and cultured as previously described .
The pRF vectors containing FMDV-IRES (serotype C) , EMCV-IRES, and HCV-IRES  were kind gifts from Dr. Hirasawa and Professor Sung-Key Jang. The pCAGGS-Neo vector was constructed using pCAG Neo (Fujifilm Wako, Tokyo, Japan) and pCAGGS vectors (cat. no. RDB08938; Riken Bank, Ibaraki, Japan). Reporter genes were excised from pCAGGS/FMDV-IRES  using the restriction endonucleases EcoRV (Toyobo, Osaka, Japan) and BamHI (New England Biolabs, Ipswich, MA, USA). The pCAGGS-Neo/FMDV-IRES vector was generated by inserting a reporter gene into pCAGGS-Neo, which was then treated with EcoRV (Toyobo), BamHI (New England Biolabs), and rAPid Alkaline Phosphatase (Roche, Basel, Switzerland) using Mighty Mix (Takara, Shiga, Japan).
The FMDV-IRES short hairpin RNA (shRNA) expression vector was constructed using the pLL3.7 vector (cat. no. 11795; Addgene, Watertown, MA, USA). The shRNA sequence (5′-tACAGGCTAAGGATGCCCTTCAGGTAttcaagagaTACCTGAAGGGCATCCTTAGCCTGTttttttC-3′, where capital letters indicate the target sequence and lower-case letters indicate the loop region) was subcloned under the U6 promoter.
DNA sequencing was performed by FASMAC Co. (Kanagawa, Japan), and DNA sequence characterization was performed using GENETYX-Mac software (GENETYX Co., Tokyo, Japan) and GENBANK.
Short interfering RNA (siRNA) transfection
Northern blot analysis
Total RNA was extracted using ISOGEN (NIPPON GENE Co. Tokyo, Japan) and electrophoresed in formaldehyde agarose (1.2%) gels. Dicistronic mRNA was detected with labeled RNA using a PvuII-digested pRF vector transcribed with T7 RNA polymerase (Renilla luciferase region) and detected with digoxigenin (DIF Northern Starter kit; Merk, Darmastadt, Germany).
Transfection and lentiviral infection
Plasmid transfection was performed using Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s specifications after the cells reached 50–70% confluence. For the establishment of cell lines, HEK293 cells were cultured with medium containing G418 (300 μg/mL) after transfection with the pCAGGS-Neo/FMDV-IRES vector. After 3–4 weeks, G418-resistant cells were identified as colonies. siRNA (1, 5, or 10 nM) reverse transfection was performed using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s specifications. Lentiviral packaging was performed using MISSION Lentiviral Packaging Mix (Sigma-Aldrich, St. Louis, MO, USA), and infection with lentivirus was performed according to the manufacturer’s instructions. Titration of lentivirus was performed via detection of green fluorescent protein (GFP) using a fluorescence microscope (Bz-x700; Keyence, Osaka, Japan). Cell viability was measured using WST assays (Dojindo, Kumamoto, Japan) by determining the optical density at 450 nm (OD450), according to the manufacturer’s instructions. Luciferase assays were performed using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Luminescence was measured with a GloMax 96 Microplate Luminometer (Promega) for 10 s, as previously described .
All data are presented as means ± standard deviations from three independent experiments. Statistical analysis was performed using multiple t tests corrected for multiple comparisons using the Holm–Sidak method (Graph Pad Prism ver. 8.1.2) to evaluate significant differences. Results with P values of less than 0.05 were considered significant.
Identification of the conserved region among FMVD-IRES sequences and design of the siRNA sequence
Establishment of FMDV-IRES-expressing cell lines
In order to evaluate the efficacy of siRNA-mediated FMDV-IRES silencing, we established cell lines containing a bicistronic reporter plasmid . Using EcoRV and BamHI, a fragment was excised from the pCAGGS/FMDV-IRES vector , which contained an FMDV-IRES element  between the Renilla and firefly luciferase genes. This fragment was then inserted into the pCAGGS-Neo/MCS vector. The resulting plasmid construct was named pCAGGS-Neo/FMDV-IRES (Supplementary Fig. 2).
Effects of siRNA on IRES-mediated translation of FMDV
Establishment of an shRNA expression vector targeting the FMDV-IRES conserved region
To sustain the effects of silencing, we constructed an shRNA expression vector utilizing a lentiviral expression vector (Supplementary Fig. 3A). We subcloned the shRNA sequence under the control of the U6 promoter. Transfection with the shRNA expression vector was confirmed with fluorescence microscopy using the GFP gene encoded in this vector (Supplementary Fig. 3A, B).
In this study, we evaluated the silencing effects of siRNA and shRNA targeting a conserved region of the FMDV-IRES among the seven FMDV serotypes. The lenti-shRNA expression vector generated shRNA under control of the U6 promoter persistently , enabling observation of its suppressive effects on FMDV-IRES-mediated translational activity in HEK293 cells after 14 days without significant cytotoxicity. These features make this construct promising in terms of solving several current challenges with FMDV vaccination. Moreover, this lenti-shRNA expression vector could be applicable to transduction in embryos for construction of transgenic animals . For establishment of therapeutic vectors in vivo in future studies, it will be necessary to use vectors other than lentiviruses, e.g., adenovirus vectors [22, 23] and adeno-associated virus vectors .
FMDV populations show high levels of genetic diversity, mainly because of the lack of RNA polymerase proofreading ability. This diversity makes disease control using vaccines and laboratory diagnosis difficult . For example, because of antigenic diversity between serotypes and genotypes, vaccination with another serotype or genotype of the same serotype may fail to control disease . In addition, new variant viruses generated after vaccination through escape mutation can cause vaccine failure . However, for the IRES element, the nucleotide sequence is relatively more conserved than that of viral structural proteins. Because IRES function is supported by higher-order RNA structures, including stem-loop structures, disruption of these structures decreases IRES activity. Thus, IRES mutants do not replicate efficiently, selecting for relative conservation of the IRES region . Moreover, the IRES conserved region could be a target for prevention of FMDV infection [28, 29]. This makes the IRES region a suitable target for pan-serotype antiviral drugs for FMDV. However, there is also a risk for generating resistant escape mutant viruses , and this should be evaluated in future studies.
The efficacies of vaccines and drugs can be influenced by the host animal species. For example, levels of FMDV replication are significantly higher in pigs than in other animals , which may be related to the influence of host factors on RNA replication. However, for IRES-mediated translation, activity is not significantly influenced by the cell line origin , and host translation factors (e.g., eIF4E, eIF2, and eIF3) are highly conserved among animal species. Therefore, FMDV-IRES shRNA is expected to be effective in all animal species.
FMDV is a highly contagious agent that can be studied only in special facilities with highly regulated biosecurity protocols . The FMDV-IRES-expressing cells established in this study could enable the screening of new inhibitors of FMDV replication in laboratories with less stringent security clearance.
In summary, the IRES-mediated translational activity of FMDV may be a suitable target for the development of pan-serotype antiviral drugs because of the relatively high sequence conservation of IRES among FMDV serotypes. The FMDV-IRES shRNA-expressing vector and FMDV-IRES-expressing cells established in this study provide new tools for the screening of anti-FMDV drugs. Future studies to evaluate antiviral effects and improve the shRNA delivery vector system are required for establishment of anti-FMDV drugs.
The authors thank Dr. D. Yamane for his valuable comments on the experimental protocols. This study was supported by a grant from the Ministry of Education, Science and Culture, Japan (Grant No. 15K14781).
TM, YH, TK, and KTK performed experiments; KTK designed the experiment and wrote the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This study was performed in accordance with institutional committee protocols of Kagoshima University.
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