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Abstract
BackgroundEbola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized.Methodology/Principal findingsUsing multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication.Conclusions/SignificanceWe have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target regulating an essential aspect of the EBOV life cycle.
Highlights
Ebola virus (EBOV) is a member of the genus Ebolavirus in the family of Filoviridae, and the causative agent of a severe hemorrhagic fever called Ebola virus disease (EVD) with case fatality rates of up to 90% [1]
Until now no experimental evidence has been provided identifying the catalytic center of the viral RNA-dependent RNA polymerase, which is absolutely essential for the virus life cycle due to its role in replicating and transcribing the viral negative-sense RNA genome
Based on a comparison to related negative-sense RNA viruses from other virus families we identified a putative catalytic center within the Ebola virus polymerase, and provide the experimental evidence that the Ebola virus polymerase utilizes a classical GDNQ motif for both genome replication and transcription
Summary
Ebola virus (EBOV) is a member of the genus Ebolavirus in the family of Filoviridae, and the causative agent of a severe hemorrhagic fever called Ebola virus disease (EVD) with case fatality rates of up to 90% [1]. A number of experimental therapeutics are under development, many of which target the viral polymerase L (reviewed in [4,5]) This viral protein acts in concert with the other viral ribonucleoprotein complex (RNP) proteins, the nucleoprotein NP, the polymerase cofactor VP35, and the transcriptional activator VP30, to facilitate replication of the negative sense RNA genome of EBOV, as well as its transcription into viral mRNAs [6]. A miniature version of the viral genome (a so-called minigenome), in which all viral open reading frames have been removed and replaced by a reporter gene, but in which the non-coding terminal leader and trailer regions are retained, is expressed together with the RNP proteins in mammalian cells These RNP proteins recognize the minigenome as an authentic viral template based on its leader and trailer regions, and replicate and transcribe it resulting in reporter activity levels that mirror these steps in the viral life cycle. Until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized
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