Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 20132016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP\u27s roles in protein structure, virion budding, and transcription and replication

Interfering with nucleotide excision by the coronavirus 3′-to-5′ exoribonuclease

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3′-to-5′ proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3′-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3′-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible

The Marburgvirus-Neutralizing Human Monoclonal Antibody MR191 Targets a Conserved Site to Block Virus Receptor Binding

Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%–90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CX_6CC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific “wing” antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure

Assembly of the Ebola Virus Nucleoprotein from a Chaperoned VP35 Complex

Ebolavirus NP oligomerizes into helical filaments found at the core of the virion, encapsidates the viral RNA genome, and serves as a scaffold for additional viral proteins within the viral nucleocapsid. We identified a portion of the phosphoprotein homolog VP35 that binds with high affinity to nascent NP and regulates NP assembly and viral genome binding. Removal of the VP35 peptide leads to NP self-assembly via its N-terminal oligomerization arm. NP oligomerization likely causes a conformational change between the NP N- and C-terminal domains, facilitating RNA binding. These functional data are complemented by crystal structures of the NP°-VP35 complex at 2.4 Å resolution. The interactions between NP and VP35 illuminated by these structures are conserved among filoviruses and provide key targets for therapeutic intervention

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

<div><p>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 <i>in vitro</i> 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.</p></div

Analysis of RNA levels shows the VP30-NP interaction to be a general modulator of RNA synthesis activity.

<p>RNA from mini-replicon transfected cells was reverse transcribed using primers specific for vRNA (blue), cRNA (red) or mRNA (green). Quantitative PCR was performed with primers amplifying the firefly luciferase ORF common to all RNA types. VP30 mutants are indicated and are arranged from high affinity (left) to low affinity (right) binders. Wild-type (WT) and negative controls (-L, -3E5E-ffLuc, -DNA) are presented for comparison. Data are normalized to the wild-type control samples.</p

EBOV VP30 mutations to the NP-binding site alter the affinity of the interaction.

<p>EBOV VP30 mutations to the NP-binding site alter the affinity of the interaction.</p

The NP 600–620 peptide is essential for mini-replicon activity.

<p>Mammalian cells were transfected with plasmids encoding the Ebola virus RNA synthesis components NP, VP35, VP30 and L, along with a model viral mini-replicon encoding firefly luciferase. Full-length NP was exchanged with either NP 1–600 or 1–620 in the presence or absence of VP30. A full-length NP mutant, in which the conserved residues from 605–612 had been scrambled to 605-PVARPYAP-612, was also included. Negative controls for the assay included transfections lacking the viral polymerase (-L) or the mini-replicon RNA (-3E5E-ffLuc) and a no-DNA mock transfection (-DNA). A) Data is shown as the percent luciferase activity compared to the wild-type positive control (left-most sample) and is the average of three independent experiments. B) Western blot for NP, VP35 and VP30 of cleared cellular lysates transfected with the mini-replicon components. All truncated and mutant NPs expressed at levels similar to wild-type NP.</p

The gradient of VP30-NP interaction affinities shows two phases of RNA synthesis activity.

<p>Increasing affinity (decreasing K_D) of the VP30-NP interaction (blue) from wild-type (WT), as assessed by multiple assays (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.t002" target="_blank">Table 2</a>, Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.g004" target="_blank">4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.g007" target="_blank">7</a>), represses viral RNA synthesis activity (pink) (Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.g008" target="_blank">8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005937#ppat.1005937.g009" target="_blank">9</a>) while mildly decreasing affinity activates viral RNA synthesis. A transition occurs beyond the affinity of the VP30 D202R mutant (dashed line) such that further reductions in affinity result in diminished RNA synthesis activity reflecting the essential nature of the VP30-NP interaction to the RNA synthesis complex.</p

Mutation of the VP30 NP-binding site alters replicase activity.

<p>A) Mini-replicon activities are presented as a percentage of the firefly luciferase activity observed with wild-type VP30. Dissociation constants observed in ITC experiments are listed for each mutant. B) Western blot for NP, VP35 and VP30 of cleared lysates from cells transfected with the mini-replicon system, confirming similar levels of expression compared for VP30 mutants to wild-type.</p