Filoviruses, which include Ebola virus (EBOV) and Marburg virus, are negative-sense RNA viruses associated with sporadic outbreaks of severe viral hemorrhagic fever characterized by uncontrolled virus replication. The extreme virulence and emerging nature of these zoonotic pathogens make them a significant threat to human health. Replication of the filovirus genome and production of viral RNAs require the function of a complex of four viral proteins, the nucleoprotein (NP), viral protein 35 (VP35), viral protein 30 (VP30) and large protein (L). The latter performs the enzymatic activities required for production of viral RNAs and capping of viral mRNAs. Although it has been recognized that interactions between the virus-encoded components of the EBOV RNA polymerase complex are required for viral RNA synthesis reactions, specific molecular details have, until recently, been lacking. New efforts have combined structural biology and molecular virology to reveal in great detail the molecular basis for critical protein-protein interactions (PPIs) necessary for viral RNA synthesis. These efforts include recent studies that have identified a range of interacting host factors and in some instances demonstrated unique mechanisms by which they act. For a select number of these interactions, combined use of mutagenesis, over-expressing of peptides corresponding to PPI interfaces and identification of small molecules that disrupt PPIs have demonstrated the functional significance of virus-virus and virus-host PPIs and suggest several as potential targets for therapeutic intervention.

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 analyses revealed that 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.

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.