Ebola virus VP30 and nucleoprotein interactions modulate viral RNA synthesis

Wei Xu,Daisy W Leung,Chao Wu,Christopher F Basler,Gaya K Amarasinghe,Jyoti Batra,Priya Luthra

doi:10.1038/ncomms15576

Abstract

Ebola virus (EBOV) is an enveloped negative-sense RNA virus that causes sporadic outbreaks with high case fatality rates. Ebola viral protein 30 (eVP30) plays a critical role in EBOV transcription initiation at the nucleoprotein (eNP) gene, with additional roles in the replication cycle such as viral assembly. However, the mechanistic basis for how eVP30 functions during the virus replication cycle is currently unclear. Here we define a key interaction between eVP30 and a peptide derived from eNP that is important to facilitate interactions leading to the recognition of the RNA template. We present crystal structures of the eVP30 C-terminus in complex with this eNP peptide. Functional analyses of the eVP30–eNP interface identify residues that are critical for viral RNA synthesis. Altogether, these results support a model where the eVP30–eNP interaction plays a critical role in transcription initiation and provides a novel target for the development of antiviral therapy.

Highlights

Ebola virus (EBOV) is an enveloped negative-sense RNA virus that causes sporadic outbreaks with high case fatality rates
EBOV Viral protein 30 (VP30) is critical for viral transcription, because it is required for initiation at EBOV NP, the first gene of the seven gene genome16. Ebola viral protein 30 (eVP30) has been implicated in regulation of co-transcriptional editing of viral glycoprotein mRNAs and in modulation of viral transcription reinitiation[11,17]
In order to gain molecular mechanistic insight into how eVP30/EBOV transcription initiation at the nucleoprotein (eNP) interaction promotes viral RNA synthesis, we generated a series of recombinant eVP30/eNP truncation constructs (Fig. 1a; Supplementary Fig. 1)

Summary

Results

A peptide derived from eNP binds VP30. In order to gain molecular mechanistic insight into how eVP30/eNP interaction promotes viral RNA synthesis, we generated a series of recombinant eVP30/eNP truncation constructs (Fig. 1a; Supplementary Fig. 1). PPXP motifs typically form a left-handed turn, in the eNP/eVP30 complex structures, the conformation of this motif does not form the expected polyproline type II helix Instead, these highly conserved eNP residues make extensive contacts with eVP30 residues in an extended conformation (Fig. 3a). In competition FPA, constant concentrations of FITC-eVP30BP and eVP30110–272 were incubated with increasing concentrations of unlabelled eVP30BP competitor (Supplementary Fig. 4c) Both types of experiments display a high dynamic range of 4100 and 450 arbitrary units, respectively. Using these FPAs, we tested mutants of eNP and eVP30 and identified a number of amino acid residues that showed in a minor decrease in binding when individually mutated (Fig. 4a). Using these FPAs, we tested mutants of eNP and eVP30 and identified a number of amino acid residues that showed in a minor decrease in binding when individually mutated (Fig. 4a). eNP T603A, V604A, V610A and R612A had a

Complex D

2.52 Glu197 Leu199

Methods