Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly

Abstract

Summary Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22–α23. Biochemical, biophysical, and mutational analysis revealed inter-eNP contacts within α22–α23 are critical for viral NC-assembly and regulate viral RNA synthesis. These observations suggest that the N-terminus and α22–α23 of eNP function as context dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

Cooperative changes in solvent exposure identify cryptic pockets, conformational switches, and allosteric coupling

“…3C, reveals that this exposon reports on the very same collective curling of the terminal helices into the RNA-binding cleft identified previously (53). Crucially, this dynamic process is consistent with hydrogen-deuterium exchange data that cannot be accounted for using available cryoelectron microscopy models (22). Manipulating this conformational equilibrium with small molecules or peptides could provide a powerful means of modulating the Ebola lifecycle.…”

“…As a subsequent test of our model, we assessed its ability to retrodict a conformational switch that was previously identified by Su et al (22). Proteins must frequently act as switches, altering their behavior in response to some signal.…”

“…β-Lactamase simulations were deployed on the Folding@home distributed computing platform (21), while simulations of eNP and CAP were performed on NVIDIA P100 GPUs on our local cluster. For our retrospective work, we used previously published datasets including 90.5 µs of simulation of TEM-1 β-lactamase with the M182T substitution (17), 28.0 µs of simulation of eNP (22), and 1.5 µs of simulation of CAP (23). For our prospective work on CTX-M-9 β-lactamase, we ran 76.0 μs of aggregate simulation on Folding@home.…”

“…In a second state, eNP exists as a monomer, releasing RNA to allow transcription of the viral genome (52). Recent evidence suggests that oligomerization is controlled by the curling of C-terminal helices of eNP into the RNA-binding cleft (22). We then expect an exposon to be formed by the residues in this groove and by the residues in the Cterminal tail that transiently occupies it.…”

Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication

“…These genes encode the viral nucleoprotein (NP), viral protein of 35 kDa (VP35), VP40, a secreted glycoprotein (sGP), a type I transmembrane glycoprotein (GP), a second secreted glycoprotein (ssGP), VP30, VP24, and the large protein (L), which is the viral polymerase (Kirchdoerfer et al, 2017). NP associates with the viral genomic and antigenomic RNAs throughout the course of infection and is required for viral mRNA synthesis (transcription) and viral genome RNA replication (Muhlberger, 2007; Su et al, 2018). VP35, a critical non-enzymatic cofactor for the viral RNA-dependent RNA polymerase, is required for viral RNA synthesis and also serves as a potent suppressor of innate antiviral signaling pathways (Basler et al, 2000; Cardenas et al, 2006; Leung et al, 2010; Luthra et al, 2013).…”