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Antimicrob Agents Chemother. 2010 July; 54(7): 3007–3010.
Published online 2010 April 26. doi:  10.1128/AAC.00138-10
PMCID: PMC2897319

Minigenome-Based Reporter System Suitable for High-Throughput Screening of Compounds Able To Inhibit Ebolavirus Replication and/or Transcription [down-pointing small open triangle]

Abstract

We describe an Ebolavirus minigenome-based system that is suitable for high-throughput screening of compounds able to impair Ebolavirus virus replication and/or transcription. The assay is robust (Z′ factor, >0.6) and can be carried out in low-biosafety containment. Results from a pilot screen of 960 compounds are presented.

Ebolaviruses and the closely related Marburgviruses cause hemorrhagic fever in humans and nonhuman primates, with high mortality rates (18). Currently, treatment of these infections is limited to supportive care (1). The need for efficacious therapeutics is further underscored by the increasing frequency of Ebolavirus outbreaks in both humans and endangered great ape populations (2, 20, 21).

High-throughput screening (HTS) systems should be robust with high reproducibility and should be amenable to work in low-biosafety containment. Ebolavirus is categorized as a biosafety level 4 agent, and HTS systems that rely on live Ebolavirus are thus impractical. Here, we describe a minireplicon system to screen for compounds that interfere with viral replication and/or transcription. In these systems, a virus-like RNA encoding a reporter protein is amplified by the viral proteins required for replication and transcription, i.e., the Ebolavirus polymerase (L), nucleoprotein (NP), and VP30 and VP35 proteins (10, 14).

A minireplicon system based on Zaire Ebolavirus developed in our laboratory was taken as a starting point (22). First, we generated a reporter construct (p3E5E-Luc) in which the open reading frame (ORF) for the luciferase gene (in antisense orientation) is flanked by the Zaire Ebolavirus leader and trailer regions (which contain the viral promoters for replication and transcription) and by T7 RNA promoter and terminator sequences (Fig. (Fig.11 A). Next, we transfected human embryonic kidney (293T) cells with p3E5E-Luc, pC-T7 (for T7 polymerase expression [15]), and pCEZ-NP, pCEZ-VP35, pCEZ-VP30, and pCEZ-L (for the expression of the Zaire Ebolavirus NP, VP35, VP30, and L proteins, respectively [15]) using Trans-IT LT1 reagent (Mirus); the amounts of the respective plasmids are listed in Table Table1,1, Setup number 1. To determine background expression levels of luciferase in the absence of a functional nucleocapsid complex (negative control), we substituted empty vector for pCEZ-L. Forty-eight hours posttransfection, an equal volume of Steady-Glo luciferase assay substrate (Promega) was mixed with each sample and luminescence was measured. Mean luciferase expression and standard deviations were obtained by measuring luminescence in eight separate wells for both the positive (with pCEZ-L) and negative (without pCEZ-L) controls. For this and all subsequent experiments, we next calculated the Z′ factor (24) as a measure of the robustness of the assay: Z′ = 1 − [3 × (σp + σn)/(μpμn)], where σp is the standard deviation of the positive control (reporter protein expression from the minireplicon), σn is the standard deviation of the negative control (reporter protein expression in the absence of the L protein), μp is the mean of the positive control, and μn is the mean of the negative control. The Z′ factor incorporates the dynamic range between positive and negative controls, as well as their well-to-well variability. A Z′ factor of >0.5 is recommended for a robust screen, and values above 0.75 are preferred before transfer to a 96-well platform because the Z′ factor usually declines during adaptation to smaller formats (24).

FIG. 1.
Schematic representation of transcription cassettes for the synthesis of Ebolavirus-like RNAs. (A) Ebolavirus minigenome transcribed by T7 RNA polymerase, resulting in negative-sense, virus-like RNA. The HDV ribozyme (Rib) creates the 3′ end of ...
TABLE 1.
Plasmid cocktails used for different assay setups

In this assay, we found high well-to-well variability for the positive control and marked luciferase expression in the negative control (i.e., in the absence of the polymerase protein) (Fig. (Fig.22 A). The signal-to-background ratio was 88:1 and the Z′ factor was 0.53, which is considered too low for transfer to a HTS protocol.

FIG. 2.
Establishment of Ebolavirus minireplicon assays for HTS. Shown are results from four different assay setups. Mean luciferase expression and standard deviations were obtained by measuring luminescence from eight transfection replicates for both the positive ...

To improve the signal-to-background ratio and the Z′ factor, we next tested a minireplicon system that utilizes cellular RNA polymerase I for the transcription of virus-like RNAs (10). In this approach, the RNA polymerase used for the synthesis of virus-like RNAs is no longer provided from a plasmid, thus reducing the number of plasmids needed and thereby potentially increasing the robustness of the system.

To establish an RNA polymerase I-driven minigenome, we cloned the luciferase gene and the Ebolavirus leader and trailer regions between RNA polymerase I promoter and terminator sequences (Fig. (Fig.1B).1B). When 293T cells were transfected with this reporter construct (pHH21-3E5E-Luc; see Setup number 2 in Table Table1)1) and the indicated amounts of protein expression plasmids, the signal-to-background ratio rose to 171:1 and the Z′ factor improved to 0.80, indicating markedly reduced well-to-well variability (Fig. (Fig.2B).2B). However, appreciable background luminescence in the negative control remained a concern (Fig. (Fig.2B2B).

To determine if the use of a different cell line would result in lower background luminescence, we tested COS-1 cells. These cells, like 293T cells, constitutively express the simian virus 40 (SV40) large T antigen, which enhances protein expression levels from plasmids possessing the SV40 origin of replication as used here. Using Setup number 3 in Table Table1,1, we found that background luminescence was significantly reduced in COS-1 cells; however, well-to-well variability was increased compared to that in 293T cells, resulting in a Z′ factor of only 0.52 (Fig. (Fig.2C2C).

To further increase the robustness of the screening assay, we attempted to reduce the number of plasmids required for minigenome replication and transcription by constructing a bicistronic vector expressing NP and VP35 (pCEZ-NP-2A-NP) (Fig. (Fig.1C).1C). In this construct, a picornavirus-derived 2A-like self-cleaving peptide (5, 6, 17) separates the coding regions for NP and VP35. These viral proteins were chosen because they are required in equivalent amounts for optimal minigenome transcription (22). Next, we performed minireplicon assays with pCEZ-NP-2A-VP35 in place of pCEZ-NP and pCEZ-VP35 in COS-1 cells (Setup number 4 in Table Table1).1). The background-to-noise ratio was 7,000:1 with a Z′ factor of 0.83 (Fig. (Fig.2D),2D), indicative of a robust assay that is compatible with HTS requirements.

We transferred our optimized assay to a 96-well format in a pilot study. To simplify the screening protocol, a batch transfection procedure was used: COS-1 cells in suspension were mixed with transfection reagent-DNA complexes and mechanically plated. This protocol provides two advantages: (i) it combines cell plating and transfection into a single step, reducing the impact of pipetting errors, and (ii) it reduces the assay time by 1 day. For the pilot assay, we tested 960 well-characterized compounds from the Prestwick Chemical Known Bioactives library. Compounds suspended in dimethyl sulfoxide (DMSO) (10 μM final concentration/well) were added, and, after incubation for 32 h, luciferase expression was measured. Several wells on each plate received DMSO only (negative controls). To eliminate compounds that reduced Ebolavirus replication due to cell cytotoxicity, parallel plates were treated with the test compounds and cell viability was measured using the CellTiter-Glo luminescent cell viability assay (Promega). The percent inhibition of luciferase expression by each compound was calculated as follows: 1 − (luciferase signal of compound-treated well/mean luciferase signal of wells treated with DMSO) × 100.

Adapting the assay to the 96-well format impacted its robustness modestly (overall Z′ factor of 0.64). Six compounds were found to inhibit luciferase expression by >75% and reduce cell viability by <25% (Table (Table2).2). Cephaeline and emetine are closely related alkaloid components of ipecac (19) (a well-known substance used to induce vomiting), and emetine has been shown to inhibit protein synthesis and promote apoptosis in mammalian cells (3, 9, 13). Emetine appears to be an especially potent inhibitor of translation of viral mRNA in some systems (16). Patulin and salinomycin have antibacterial properties and are also proapoptotic compounds (7, 12, 23). It has been shown that patulin can specifically inhibit replication of a fungal double-stranded RNA (dsRNA) virus (4). Doxorubicin and ellipticine appear to suppress DNA replication in eukaryotic cells through several mechanisms, including DNA intercalation and inhibition of DNA topoisomerase II activity (8, 25). Doxorubicin can also block Tat-mediated enhancement of HIV long terminal repeat (LTR)-driven transcription by recruiting suppressive nuclear factors to the transactivation response element of the HIV promoter (11). The relative impact of these compounds on COS-1 cell metabolism and architecture versus Ebolavirus minigenome replication, as well as live Ebolavirus replication, is currently under investigation, and additional compound libraries are being tested.

TABLE 2.
Compounds that inhibit reporter gene expression by >75% and reduce cell viability by <25%

In conclusion, the Ebolavirus minigenome-based assay described here enables rapid screening of compounds for inhibitors of Ebolavirus replication and/or transcription.

Acknowledgments

We thank Noel Peters and Lane Milde at the Keck-UWCCC Small Molecule Screening Facility on the University of Wisconsin-Madison campus for their assistance with compound screening.

Support for this work came from National Institute of Allergy and Infectious Diseases and Public Health Service research grants.

Footnotes

[down-pointing small open triangle]Published ahead of print on 26 April 2010.

REFERENCES

1. Bausch, D. G., H. Feldmann, T. W. Geisbert, M. Bray, A. G. Sprecher, P. Boumandouki, P. E. Rollin, C. Roth, and the Winnipeg Filovirus Clinical Working Group. 2007. Outbreaks of filovirus hemorrhagic fever: time to refocus on the patient. J. Infect. Dis. 196(Suppl. 2):S136-S141. [PubMed]
2. Bermejo, M., J. D. Rodriguez-Teijeiro, G. Illera, A. Barroso, C. Vila, and P. D. Walsh. 2006. Ebola outbreak killed 5000 gorillas. Science 314:1564. [PubMed]
3. Boon-Unge, K., Q. Yu, T. Zou, A. Zhou, P. Govitrapong, and J. Zhou. 2007. Emetine regulates the alternative splicing of Bcl-x through a protein phosphatase 1-dependent mechanism. Chem. Biol. 14:1386-1392. [PMC free article] [PubMed]
4. Detroy, R. W., and P. E. Still. 1976. Patulin inhibition of mycovirus replication in Penicillium stoloniferum. J. Gen. Microbiol. 92:167-174. [PubMed]
5. Donnelly, M. L., L. E. Hughes, G. Luke, H. Mendoza, E. ten Dam, D. Gani, and M. D. Ryan. 2001. The “cleavage” activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring “2A-like” sequences. J. Gen. Virol. 82:1027-1041. [PubMed]
6. Donnelly, M. L., G. Luke, A. Mehrotra, X. Li, L. E. Hughes, D. Gani, and M. D. Ryan. 2001. Analysis of the aphthovirus 2A/2B polyprotein “cleavage” mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal “skip.” J. Gen. Virol. 82:1013-1025. [PubMed]
7. Fuchs, D., A. Heinold, G. Opelz, V. Daniel, and C. Naujokat. 2009. Salinomycin induces apoptosis and overcomes apoptosis resistance in human cancer cells. Biochem. Biophys. Res. Commun. 390:743-749. [PubMed]
8. Garbett, N. C., and D. E. Graves. 2004. Extending nature's leads: the anticancer agent ellipticine. Curr. Med. Chem. Anticancer Agents 4:149-172. [PubMed]
9. Grollman, A. P. 1966. Structural basis for inhibition of protein synthesis by emetine and cycloheximide based on an analogy between ipecac alkaloids and glutarimide antibiotics. Proc. Natl. Acad. Sci. U. S. A. 56:1867-1874. [PubMed]
10. Groseth, A., H. Feldmann, S. Theriault, G. Mehmetoglu, and R. Flick. 2005. RNA polymerase I-driven minigenome system for Ebola viruses. J. Virol. 79:4425-4433. [PMC free article] [PubMed]
11. Jeyaseelan, R., M. Kurabayashi, and L. Kedes. 1996. Doxorubicin inhibits Tat-dependent transactivation of HIV type 1 LTR. AIDS Res. Hum. Retroviruses 12:569-576. [PubMed]
12. Liu, B. H., T. S. Wu, F. Y. Yu, and C. H. Wang. 2006. Mycotoxin patulin activates the p38 kinase and JNK signaling pathways in human embryonic kidney cells. Toxicol. Sci. 89:423-430. [PubMed]
13. Moller, M., K. Herzer, T. Wenger, I. Herr, and M. Wink. 2007. The alkaloid emetine as a promising agent for the induction and enhancement of drug-induced apoptosis in leukemia cells. Oncol. Rep. 18:737-744. [PubMed]
14. Muhlberger, E., M. Weik, V. E. Volchkov, H. D. Klenk, and S. Becker. 1999. Comparison of the transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems. J. Virol. 73:2333-2342. [PMC free article] [PubMed]
15. Neumann, G., H. Feldmann, S. Watanabe, I. Lukashevich, and Y. Kawaoka. 2002. Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture. J. Virol. 76:406-410. [PMC free article] [PubMed]
16. Ramabhadran, T. V., and R. E. Thach. 1980. Specificity of protein synthesis inhibitors in the inhibition of encephalomyocarditis virus replication. J. Virol. 34:293-296. [PMC free article] [PubMed]
17. Ryan, M. D., and M. Flint. 1997. Virus-encoded proteinases of the picornavirus super-group. J. Gen. Virol. 78(part 4):699-723. [PubMed]
18. Sanchez, A., T. W. Geisbert, and H. Feldmann. 2007. Filoviridae: Marburg and ebola viruses, p. 1409-1448. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 5th ed., vol. 1. Lippincott Williams & Williams, Philadelphia, PA.
19. Scharman, E. J., J. M. Hutzler, J. G. Rosencrance, and T. S. Tracy. 2000. Single dose pharmacokinetics of syrup of ipecac. Ther. Drug Monit. 22:566-573. [PubMed]
20. Towner, J. S., T. K. Sealy, M. L. Khristova, C. G. Albarino, S. Conlan, S. A. Reeder, P. L. Quan, W. I. Lipkin, R. Downing, J. W. Tappero, S. Okware, J. Lutwama, B. Bakamutumaho, J. Kayiwa, J. A. Comer, P. E. Rollin, T. G. Ksiazek, and S. T. Nichol. 2008. Newly discovered ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog. 4:e1000212. [PMC free article] [PubMed]
21. Walsh, P. D., K. A. Abernethy, M. Bermejo, R. Beyers, P. De Wachter, M. E. Akou, B. Huijbregts, D. I. Mambounga, A. K. Toham, A. M. Kilbourn, S. A. Lahm, S. Latour, F. Maisels, C. Mbina, Y. Mihindou, S. N. Obiang, E. N. Effa, M. P. Starkey, P. Telfer, M. Thibault, C. E. Tutin, L. J. White, and D. S. Wilkie. 2003. Catastrophic ape decline in western equatorial Africa. Nature 422:611-614. [PubMed]
22. Watanabe, S., T. Watanabe, T. Noda, A. Takada, H. Feldmann, L. D. Jasenosky, and Y. Kawaoka. 2004. Production of novel Ebola virus-like particles from cDNAs: an alternative to Ebola virus generation by reverse genetics. J. Virol. 78:999-1005. [PMC free article] [PubMed]
23. Wu, T. S., Y. C. Liao, F. Y. Yu, C. H. Chang, and B. H. Liu. 2008. Mechanism of patulin-induced apoptosis in human leukemia cells (HL-60). Toxicol. Lett. 183:105-111. [PubMed]
24. Zhang, J. H., T. D. Chung, and K. R. Oldenburg. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4:67-73. [PubMed]
25. Zunino, F., and G. Capranico. 1990. DNA topoisomerase II as the primary target of anti-tumor anthracyclines. Anticancer Drug Des. 5:307-317. [PubMed]

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