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Sci Rep. 2018 Mar 5;8(1):3970. doi: 10.1038/s41598-018-22328-3.

Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus.

Author information

1
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
2
Li Ka Shing Institute of Virology at University of Alberta, Edmonton, Alberta, Canada.
3
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada. gotte@ualberta.ca.
4
Li Ka Shing Institute of Virology at University of Alberta, Edmonton, Alberta, Canada. gotte@ualberta.ca.

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.

PMID:
29507309
PMCID:
PMC5838098
DOI:
10.1038/s41598-018-22328-3
[Indexed for MEDLINE]
Free PMC Article

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