Dataset Information

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.

SUBMITTER: Nelson EV

PROVIDER: S-EPMC5654650 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Nelson Emily V EV Pacheco Jennifer R JR Hume Adam J AJ Cressey Tessa N TN Deflubé Laure R LR Ruedas John B JB Connor John H JH Ebihara Hideki H Mühlberger Elke E

Antiviral research 20170812

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 us ...[more]

PMID: 28807685

Similar Datasets

Project description:Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available, which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL4) laboratories. The EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL2 conditions. Here, we developed a novel EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The minigenomic RNA harboring a Gaussia luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and L proteins. Quantification of viral RNA (vRNA), cRNA, and mRNA levels of the EBOV minigenome demonstrated that the stable EBOV replicon had much-more-active minigenome replication than previously developed transient-transfection-based EBOV minigenome systems, which recapitulate viral primary transcription more than genome replication. Interestingly, minigenome replication in the stable EBOV replicon cells was insensitive to interferon treatment or RNA interference. Moreover, RNase digestion of the replicon cell lysates revealed the remarkably stable nature of the EBOV minigenomic vRNA ribonucleoprotein complex, which may help improve understanding of EBOV persistence in convalescent patients.IMPORTANCE The scope and severity of the recent Ebola outbreak in Western Africa justified a more comprehensive investigation of the causative risk group 4 agent Ebola virus (EBOV). Study of EBOV replication and antiviral development can be facilitated by developing a cell culture system that allows experimentation under biosafety level 2 conditions. Here, we developed a novel stable EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The replicon system had more-active genome replication than previously developed transient-transfection-based EBOV minigenome systems, providing a convenient surrogate system to study EBOV replication. Furthermore, self-replicating minigenomic vRNA in the replicon cells displayed strong stability in response to interferon treatment, RNA silencing, and RNase digestion, which may provide an explanation for the persistence of EBOV in survivors.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets