Minigenome-Based Reporter System Suitable for High-Throughput Screening of Compounds Able To Inhibit Ebolavirus Replication and/or Transcription

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. 1A). 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 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 ZЈ factor (24) as a measure of the robustness of the assay:, where p is the standard deviation of the positive control (reporter protein expression from the minireplicon), n is the standard deviation of the negative control (reporter protein expression in the absence of the L protein), p is the mean of the positive control...

Assembly of the Ebola Virus Nucleoprotein from a Chaperoned VP35 Complex

“…Mutated VP35 1-80 was used in ITC analysis, while fulllength VP35 mutants were used in Ebola virus mini-genome assays. While the ITC assays directly measure the strength of the interaction between the NP° core and VP35 N-terminal peptide, the mini-genome assay assesses the abilities of these mutants in the background of full-length viral proteins in mammalian cells to support replication/transcription of a reporter gene whose expression is controlled by viral sequences (Jasenosky et al, 2010). Selected VP35 hydrophobic amino acids were mutated to aspartate to disrupt hydrophobic interactions.…”

“…Screening of VP35 mutants was done as in (Jasenosky et al, 2010). HEK 293T cells were transfected with plasmids expressing NP, VP35, VP30, L, T7 RNA polymerase, Renilla luciferase proteins and the gene for firefly luciferase flanked by viral sequences whose expression is driven by a T7 RNA polymerase promoter.…”

The Cytoprotective Enzyme Heme Oxygenase-1 Suppresses Ebola Virus Replication

“…To further examine the mechanism underlying CoPP-dependent inhibition of EBOV growth, we tested the effects of CoPP treatment and HO-1 expression on EBOV transcription and replication in a luciferase-based EBOV minireplicon system. To generate virus-like RNA, 293T cells were transfected with an EBOV minireplicon plasmid encoding firefly luciferase under the control of the RNA polymerase I promoter, along with plasmids encoding EBOV proteins required for transcription and replication, i.e., NP, VP30, VP35, and L (31). To test the effect of CoPP treatment, cells were washed 6 h posttransfection and treated with 10 M CoPP or 10 M SnPP for 48 h, after which cell lysates were harvested and assessed for luciferase activity.…”

“…To examine viral transcription/replication, a luciferase activity-based EBOV minireplicon assay was performed as described previously (31). Briefly, 293T cells or HEK 293 VP30/HO-1 cells were seeded into 12-well tissue culture plates and transfected with the following EBOV protein expression plasmids: pCAGGS EBOV NP (0.75 g), pCAGGS EBOV VP35 (0.…”

Against the clock towards new Ebola virus therapies

“…Several published studies have used an Ebolavirus minigenome-based platform that allows highthroughput screening (HTS) analysis to find compounds able to impair viral replication or transcription (Jasenosky et al, 2010;Watanabe et al, 2004). This assay makes HTS system possible, since it can be carried out in biocontainment conditions less strict than BSL-4.…”