Research Article: The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

Date Published: October 18, 2016

Publisher: Public Library of Science

Author(s): Robert N. Kirchdoerfer, Crystal L. Moyer, Dafna M. Abelson, Erica Ollmann Saphire, Matthias Johannes Schnell.

http://doi.org/10.1371/journal.ppat.1005937

Abstract

Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.

Partial Text

Filoviruses such as Ebola (EBOV) and Marburg viruses (MARV) are nonsegmented negative-sense RNA viruses that can cause deadly hemorrhagic fevers with up to 90% fatality [1]. The impact of EBOV is highlighted by the recent outbreak in West Africa involving over 28,000 cases and claiming more than 11,000 lives [2]. Key to the viral life cycle are the components of the viral nucleocapsid. The nucleocapsids of all nonsegmented negative-sense RNA viruses carry a viral RNA-dependent, RNA polymerase (L), a phosphoprotein polymerase co-factor (P or VP35) and a nucleoprotein (N or NP), which encapsidates the viral genome. In the Mononegavirales order of viruses, L and NP interact through the phosphoprotein to carry out viral RNA synthesis. Filoviruses are unusual among mononegaviruses in that they encode an additional nucleocapsid component, VP30.

In this work, we have identified the region of filovirus NPs necessary and sufficient for interaction with VP30. We broadened our initial screening for the EBOV NP-VP30 interaction to confirm that this interaction also occurs in SUDV and MARV, highlighting the conserved nature of the NP-VP30 interaction and suggesting an important role for this interaction in the filovirus life cycles. Our crystal structures of the EBOV and MARV NP-VP30 complexes have revealed the molecular determinants of these interactions and have allowed us to structurally design mutants specifically targeting the VP30 and NP interfaces.

 

Source:

http://doi.org/10.1371/journal.ppat.1005937

 

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