Virion, nucleocapsid, and inclusion body structures of Lloviu and Ebola viruses

Virion, nucleocapsid, and inclusion body structures of Lloviu and Ebola viruses Structural diversity of filovirus virions and in particular nucleocapsids has been observed between Ebola virus (EBOV) and Marburg virus (MARV). Lloviu virus (LLOV), a newly discovered filovirus, sits phylogenetically between EBOV and MARV, providing a rare opportunity to expand our knowledge of the biology of this dangerous family of pathogens. Our team of virology, structural biology, and bioinformatics scientists proposes to determine the morphology of LLOV virions using cryogenic electron microscopy and tomography (cryo-EM, cryo-ET), and compare the structures of its nucleocapsid and its surface glycoproteins to those of EBOV and MARV. In addition, our proposed studies will provide insights into the replication of LLOV and EBOV by determining three-dimensional structures of viral replication factories (also called 'inclusion bodies') in infected human cells using thick sections (~160 nm) of high pressure frozen/freeze-substituted samples for tomography. Thin sections (~50 nm) will be analyzed using immunolabeling of viral proteins and RNA in-situ hybridization to localize sites of viral transcription, genome replication, and nucleocapsid assembly. Our studies will generate three-dimensional structures of LLOV virions, including structures of isolated viral nucleocapsids. Macromolecular structures of virus nucleocapsids and virions can provide critical data with regards to protein-protein interaction domains that could serve as the targets of small molecule inhibitors. In addition, electron tomography will provide novel insights into the spatio-temporal morphology of viral replication factories within LLOV- and EBOV-infected cells. In the proposed studies we will combine our unique expertise in handling BSL-4 pathogens with our deep experience in structural biology to generate foundational knowledge with respect to filovirus replication.