Ebola virus is among the deadliest viruses known and caused an outbreak of unprecedented scale in 2014. Much of how the virus replicates, transcribes and assembles remain poorly understood, in part, because no structural models yet exist by which we may interpret these processes. Ebola virus belongs to the filovirus family, which are single-stranded, negative-sense RNA viruses. In the filoviruses, the RNA genome is encapsidated by copies of the nucleoprotein NP, which assemble about the genome into an organized helical complex. Bound to copies of NP are additional viral proteins termed VP35, VP30, VP24, and L. Together, this complex is called the nucleocapsid, and it forms the macromolecular machine by which the genome is replicated and proteins are transcribed. The entire nucleocapsid complex is packaged into budding virions by binding of NP to the viral matrix protein VP40. Understanding the structures, protein-protein interactions, and assemblies of proteins in this complex is key to development of antivirals. However, no structures of NP by which we may interpret these functions yet exist - because it has previously been impossible to produce samples suitable for crystallization and high-resolution electron microscopy. Indeed, existing models of nucleocapsids are low in resolution and do not agree with each other. After several years of effort, we have succeeded in producing homogeneous, monodisperse Ebola virus NP that crystallizes and diffracts to 2.4. These crystals will be used in Specific Aim 1 to provide the first crystal structure of Ebola virus NP by which we may interpret its RNA-binding and replication functions.
Specific Aim 2 will use straightforward biochemistry to map the minimal binding domains of NP for each of its nucleocapsid binding partners. One interaction has already been identified and has resulted in 3.0 diffraction of the complex.
In Specific Aim 3, we aim to use these high-quality samples to assemble and image nucleocapsid complexes, using a stepwise in vitro assembly strategy for unambiguous assignment of density to proteins. We are aided in this effort by our state-of-the-art instrumentation and a battery of in vitro and cellular assays to confirm the biological relevance of each sample in our analysis. The resulting images of NP alone and in its assembled complexes will provide transformative roadmaps for functional analysis and medical defense.
Ebola virus can be up to 90% lethal and caused a 2014 outbreak of unprecedented scale. This project will provide molecular images of the essential viral nucleocapsid, will illuminate how the virus assembles and replicates and will provide blueprints for development of antiviral drugs against it.
| Kirchdoerfer, Robert N; Cottrell, Christopher A; Wang, Nianshuang et al. (2016) Pre-fusion structure of a human coronavirus spike protein. Nature 531:118-21 |
| Kirchdoerfer, Robert N; Abelson, Dafna M; Li, Sheng et al. (2015) Assembly of the Ebola Virus Nucleoprotein from a Chaperoned VP35 Complex. Cell Rep 12:140-149 |
