An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening

Abstract

Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research.

Keywords: Antiviral drug screening; Ebola virus; Filoviruses; Minigenome system; RNA polymerase II; T7 RNA polymerase.

Figure 1. Cloning of the pol II EBOV minigenome in pCAGGS

The minigenome sequence is flanked by two ribozymes. HH rib, hammerhead ribozyme; HDV rib, hepatitis delta ribozyme. Transcription of the minigenome by pol II leads to the production of negative-sense minigenomes. The EBOV leader and trailer regions are shown as dark gray boxes. The gene start signal is shown as a white triangle and the gene end (GE) signal as a white box. eGFP is shown as an example reporter gene in the green box. eGFP is flanked by the 3′ UTR of NP (negative sense) and the 5′ UTR of L (negative sense) as shown in light gray boxes (not to scale).

Figure 2. Comparison of the T7 and pol II eGFP minigenome systems

293T cells were transfected with the indicated concentrations of either the T7 (A) or pol II (B) driven minigenome system along with the necessary support plasmids. As a negative control, cells were co-transfected with a plasmid encoding inactive L (L_synth−) in place of the plasmid encoding the functional L. Images are representative of eGFP expression (shown in green) monitored at one, two, and three days post transfection (DPT) from two independent experiments.

Figure 3. Comparison of the T7 and Pol II luciferase minigenome systems

293T cells were transfected with 50 ng (A), 250 ng (B), or 750 ng (C) of either the T7 or pol II promoter driven minigenome plasmids containing the firefly luciferase reporter gene along with the necessary support plasmids. As a negative control, cells were co-transfected with a plasmid encoding inactive L (L_synth−) in place of the functional L plasmid. Luciferase activity of cells transfected with the L_synth− mutant was set as background activity. As a transfection efficiency control, the cells were also transfected with pMIR β-gal. Luciferase activity was normalized to beta-galactosidase activity. Data from two independent experiments, each done in triplicate are represented as fold induction of minigenome activity (as indicated by luciferase activity) with standard error of the mean (SEM) compared to the negative control (L_synth−).

Figure 4. Comparison of pol II and T7 minigenomes in 6 different cell lines

The indicated cell lines were transfected with 750 ng of either the T7 or pol II promoter driven minigenome plasmids containing the firefly luciferase reporter gene along with the necessary support plasmids as in figure 3. Data from three independent experiments, each done in triplicate are represented as fold induction of minigenome activity (as indicated by luciferase activity) compared to the negative control (L_synth−). Significance was determined using a paired, two-tailed t test; * p < 0.05; *** p < 0.0005.

Figure 5. Identification of an EBOV replication inhibitor using the pol II minigenome system in an antiviral drug screen

293T cells were transfected with the pol II minigenome plasmid containing the firefly luciferase reporter gene and support plasmids and plated in a 96-well plate at 6 h post transfection. (A) Cells were either left untreated (nd) or were treated with JG40 at the indicated concentrations and time points post transfection. At two days post transfection, cell lysates were harvested and luciferase assays were performed. Data from three independent experiments are shown as a mean fold induction of luciferase activity divided by the negative control (expressing L_synth− instead of L), with standard error of the mean (SEM) for 6 wells for each condition. Significance was determined using a paired, two-tailed t test; * p < 0.05; ** p < 0.005. (B) Cells were transfected as in (A) but with three separate negative controls; one containing a catalytically inactive L instead of L (L_synth−), one lacking L (minus L), and one lacking VP35 (minus VP35). Data from three independent experiments are represented as in (A).