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. 2021 Jun 3;13(6):1061.
doi: 10.3390/v13061061.

M Segment-Based Minigenome System of Severe Fever with Thrombocytopenia Syndrome Virus as a Tool for Antiviral Drug Screening

Hiroshi Yamada  1 , Satoshi Taniguchi  2 , Masayuki Shimojima  2 , Long Tan  1 , Miyuki Kimura  1 , Yoshitomo Morinaga  1 , Takasuke Fukuhara  3   4 , Yoshiharu Matsuura  3   5 , Takashi Komeno  6 , Yousuke Furuta  6 , Masayuki Saijo  2 , Hideki Tani  1   7
Affiliations

M Segment-Based Minigenome System of Severe Fever with Thrombocytopenia Syndrome Virus as a Tool for Antiviral Drug Screening

Hiroshi Yamada et al. Viruses. .

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans with case fatality rates of approximately 30%. There are few treatment options for SFTSV infection. SFTSV RNA synthesis is conducted using a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are, therefore, potential antiviral targets. A library of small molecule compounds was processed using a high-throughput screening (HTS) based on an SFTSV minigenome assay (MGA) in a 96-well microplate format to identify potential lead inhibitors of SFTSV RNA synthesis. The assay confirmed inhibitory activities of previously reported SFTSV inhibitors, favipiravir and ribavirin. A small-scale screening using MGA identified four candidate inhibitors that inhibited SFTSV minigenome activity by more than 80% while exhibiting less than 20% cell cytotoxicity with selectivity index (SI) values of more than 100. These included mycophenolate mofetil, methotrexate, clofarabine, and bleomycin. Overall, these data demonstrate that the SFTSV MGA is useful for anti-SFTSV drug development research.

Keywords: SFTSV; antiviral screening; antivirals; favipiravir; minigenome assay; ribavirin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of cRNA-oriented pol I-driven minigenome. The reporter minigenome was composed of a reporter gene, eGFP, C-terminally tagged with HBT, flanked by the 3′ (leader) and 5′ (trailer) noncoding regions of SFTSV. They were flanked by hammerhead ribozyme (HamRz) sequence at the N-terminus. These transcription cassettes were then cloned between the transcriptional start and stop signals of the human RNA pol I.
Figure 2
Figure 2
Reporter gene expression from cRNA-oriented pol I-driven minigenome. 293T cells in a 12-well plate were transfected, each with 300 ng of pPolIV-SFTSV-M-eGFPHBT (MG), 300 ng of pKS-SFTSV-NP, and 150 ng of pKS-SFTSV-L plasmid per well. (A) 293T cells were transfected with the MG (a), MG and pKS-SFTSV-NP (b), MG and pKS-SFTSV-L (c), or MG, pKS-SFTSV-NP, and pKS-SFTSV-L (d) and analyzed 48 h post-transfection for expression of eGFP-HBT by fluorescence microscopy. (B) 293T cells were either transfected with empty vector (mock-transfection) or transfected with the MG, MG and pKS-SFTSV-NP (MG + NP), MG and pKS-SFTSV-L (MG + L), or MG, pKS-SFTSV-NP, and pKS-SFTSV-L (MG + NP + L). The minigenome activity of eGFP-HBT expression was assayed by measuring the luciferase activity 32 h post-transfection using the Nano-Glo HiBiT Lytic Detection System. The results shown are for three independent assays, with error bars representing standard deviations.
Figure 3
Figure 3
293T cells in a 12-well plate were transfected with 300 ng of MG plasmid along with the necessary supporting plasmids (300 ng of NP and 1500 ng of pKS-SFTSV-L) per well. (A) Time course of minigenome activity. At each time point post-transfection, luciferase activity was assessed. (B) Comparison of the minigenome activity in the three cell lines. Huh7, Vero, or BHK cells in 12-well plates were transfected with the indicated plasmids. Empty pKS336 vector was used to ensure that the total amount of DNA used in each transfection had the same minigenome activity of eGFP-HBT expression and was assayed by measuring the luciferase activity 32 h post-transfection using the Nano-Glo HiBiT Lytic Detection System (Promega, Madison, WI). (C) Effect of NSs protein on minigenome activity. 293T cells were transfected with MG, pKS-SFTSV-NP, and pKS-SFTSV-L, indicating increasing amounts of pKS-NSs or pKS336. Empty vector pKS336 was used to ensure that equal amounts of DNA were transfected into each reaction mixture. The minigenome activity was assayed by measuring the luciferase activity 32 h post-transfection. The minigenome activity was expressed as the fold induction of normalized luciferase units relative to the background control (absence of NS-expressing plasmid), corrected as a percentage of the activity observed in the presence of NP and L proteins for each MG. The results shown are for three independent assays, with error bars representing standard deviations.
Figure 4
Figure 4
The SFTSV MG assay for favipiravir or ribavirin. (A) 293T cells were transfected with MG plasmid and the necessary supporting plasmids (pKS-SFTSV-NP and pKS-SFTSV-L). At each time point post-transfection, 293T cells were seeded in a 96-well plate in media containing 640 mM of favipiravir or ribavirin. After 32 h of incubation, luciferase activities were measured. (B) Inhibitory effect of favipiravir and ribavirin on SFTSV MG activity (●) in 293T cells. One hour after transfection with MG, pKS-SFTSV-NP, and pKS-SFTSV-L plasmids, 293T cells were seeded in a 96-well plate in media containing various concentrations of favipiravir or ribavirin. After 32 h of incubation, luciferase activities were measured. MG activity is expressed as the fold induction of normalized luciferase units relative to the background control (absence of reagents), corrected as a percentage of the activity observed in the presence of NP and L proteins for each MG. Cell viability (◼) was determined in the presence of drugs using the RealTime Glo MT Cell Viability Assay in parallel. A sigmoidal dose–response curve was fitted to the data using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). The results shown are for three independent assays, with error bars representing standard deviations.
Figure 5
Figure 5
Inhibitory effect of mycophenolate mofetil, clofarabine, bleomycin, and methotrexate hydrate on SFTSV MG activity (●) in 293T cells. (A) One hour after transfection with MG, pKS-SFTSV-NP, and pKS-SFTSV-L plasmids, 293T cells were seeded in a 96-well plate in media containing various concentrations of mycophenolate mofetil (A), clofarabine (B), bleomycin (C), and methotrexate hydrate (MTX) (D). After 32 h of incubation, luciferase activities were measured. Cell viability (◼) was determined in the presence of drugs using the RealTime Glo MT Cell Viability Assay (Promega, Madison, WI) in parallel. The minigenome activity is expressed as the fold induction of normalized luciferase units relative to the background control (absence of reagents), corrected as a percentage of the activity observed in the presence of NP and L proteins for each MG. A sigmoidal dose–response curve was fitted to the data using GraphPad Prism 7. The results shown are for three independent assays, with error bars representing standard deviations.
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References

    1. Xu B., Liu L., Huang X., Ma H., Zhang Y., Du Y., Wang P., Tang X., Wang H., Kang K., et al. Metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Henan Province, China: Discovery of a new bunyavirus. PLoS Pathog. 2011;7:e1002369. doi: 10.1371/journal.ppat.1002369. - DOI - PMC - PubMed
    1. Yu X.J., Liang M.F., Zhang S.Y., Liu Y., Li J.D., Sun Y.L., Zhang L., Zhang Q.F., Popov V.L., Li C., et al. Fever with thrombocytopenia associated with a novel bunyavirus in China. N. Engl. J. Med. 2011;364:1523–1532. doi: 10.1056/NEJMoa1010095. - DOI - PMC - PubMed
    1. Kim K.H., Yi J., Kim G., Choi S.J., Jun K.I., Kim N.H., Choe P.G., Kim N.J., Lee J.K., Oh M.D. Severe fever with thrombocytopenia syndrome, South Korea, 2012. Emerg. Infect. Dis. 2013;19:1892–1894. doi: 10.3201/eid1911.130792. - DOI - PMC - PubMed
    1. Takahashi T., Maeda K., Suzuki T., Ishido A., Shigeoka T., Tominaga T., Kamei T., Honda M., Ninomiya D., Sakai T., et al. The first identification and retrospective study of Severe Fever with Thrombocytopenia Syndrome in Japan. J. Infect. Dis. 2014;209:816–827. doi: 10.1093/infdis/jit603. - DOI - PMC - PubMed
    1. Liu Q., He B., Huang S.Y., Wei F., Zhu X.Q. Severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis. Lancet Infect. Dis. 2014;14:763–772. doi: 10.1016/S1473-3099(14)70718-2. - DOI - PubMed

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