Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus

Egor P Tchesnokov,David Marchant,Parisa Raeisimakiani,Marianne Ngure,Matthias Götte

doi:10.1038/s41598-018-22328-3

Egor P Tchesnokov, David Marchant + Show 3 more

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https://doi.org/10.1038/s41598-018-22328-3

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Journal: Scientific Reports	Publication Date: Mar 5, 2018
Citations: 47	License type: open-access

Affiliation: University of Alberta

Abstract

Here we report on the expression, purification and characterization of recombinant ebola virus RNA-dependent RNA polymerase (EBOV RdRp). Active protein complexes composed of the large L protein and viral protein VP35 were isolated from insect cells and analyzed using a short primer/template substrate that allowed benchmarking against related enzymes. RNA synthesis by multiprotein complexes of EBOV, influenza B, respiratory syncytial virus (RSV) and monomeric enzymes of hepatitis C and Zika (ZIKV) viruses required a 5′-phosporylated primer. The minimum length of the primer varied between two and three nucleotides in this system. The EBOV enzyme utilizes Mg2+ as a co-factor and the D742A substitution provides an active site mutant that likely affects binding of the catalytic metal ions. Selectivity measurements with nucleotide analogues translate our assay into quantitative terms and facilitate drug discovery efforts. The related EBOV and RSV enzymes are not able to efficiently discriminate against ara-cytidine-5′-triphosphate. We demonstrate that this compound acts like a non-obligate chain-terminator.

Highlights

Negative-sense RNA viruses such as influenza viruses, Measles virus, Mumps virus, respiratory syncytial virus (RSV), and ebola virus (EBOV) are important human pathogens
While the L protein is essential for RNA synthesis, the nucleoprotein NP and viral proteins VP30 and VP35 have been considered as possible additional factors[15]
RNA-dependent RNA polymerases (RdRp) of positive-sense RNA viruses such as hepatitis C virus (HCV) RdRp have been validated in this regard, and recent advances in expression and biochemical characterization of RdRp associated with negative-sense RNA viruses show promise[8,12,30]

Summary

Introduction

Negative-sense RNA viruses such as influenza viruses, Measles virus, Mumps virus, respiratory syncytial virus (RSV), and ebola virus (EBOV) are important human pathogens. Viral RNA-dependent RNA polymerases (RdRp) are essential for replication of RNA viruses and represent important drug targets. Despite recent progress in the field[4,5,6,7,8], the expression of active recombinant RdRp enzymes of negative-sense RNA viruses remains challenging. We report the expression, purification, and biochemical characterization of an active, recombinant EBOV RdRp complex. Multiprotein complexes derived from negative-sense RNA viruses RSV5 and influenza B (FluB)[6,7], and monomeric polymerases derived from positive-sense RNA viruses hepatitis C virus (HCV) and Zika virus (ZIKV) served as benchmarks[13,14]

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