Lipid-enveloped viruses replicate and bud from host cell membranes where they acquire their lipid coat. Understanding the budding processes of several viruses has had significant impact on elucidating the viral life cycle and identifying therapeutic targets. Filoviruses have a filamentous lipid- envelope and despite being discovered more than 30 years ago, not much is known on how they acquire their lipid coat. Filoviruses include Ebola virus (EBOV) and Marburg virus (MARV), which can have up to 90% clinical fatality. Filoviruses encode seven genes including the viral matrix protein VP40, which regulates budding from the host cell. VP40 as the only filovirus protein expressed in mammalian cells is sufficient to produce virus like particles (VLPs) nearly indistinguishable from live virions. Thus, VP40 has served as a model to study viral budding outside of BSL-4 laboratories. To date, little is known about how VP40 interacts with biological membranes to regulate budding and egress from the host cell plasma membrane. During the previous project period, we found that EBOV VP40 (eVP40) required PS on the inner leaflet of the plasma membrane to bind, assemble, and form VLPs. Notably, interactions of eVP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress; whereas PS is typically only on the inner leaflet. Flipping/transport of PS to the outer leaflet may aid PS presentation on the surface of nascent virions to facilitate TIM-1-mediated entry into cells. These findings provided a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. New preliminary data and collaboration with a structural biologist has demonstrated that in addition to PS, eVP40 requires plasma membrane PI(4,5)P2 for assembly and budding. In contrast, MARV VP40 (mVP40) acts as an anionic charge sensor, promiscuously binding anionic lipids consistent with the new X-ray structure solved by our collaborator. The central hypothesis is that eVP40 and mVP40 have fundamentally different lipid binding properties. This renewal proposal will investigate lipid binding, assembly, and budding properties of eVP40 and mVP40.
Specific Aim 1 will investigate the mechanisms by which eVP40 and mVP40 differentially interact with plasma membrane lipids to facilitate budding. We will also investigate the role of plasma membrane cholesterol content and membrane fluidity on eVP40 and mVP40 membrane binding, oligomerization, and budding.
Specific aim 2 will investigate the role of a plasma membrane sphingolipid in eVP40 and mVP40 VLP formation. Biochemical and cellular assays in combination with structural analysis by our collaborator will be used to elucidate the molecular architecture of the eVP40 and mVP40 assembly events. Taken together, these studies should produce important mechanistic insight into how filovirus particles form from the plasma membrane of cells.

Public Health Relevance

To date there is a lack of FDA approved treatment options or preventive measures for infections by Filoviruses, which include Ebola virus and Marburg virus. The proposed studies will provide new mechanistic insight into how the Ebola and Marburg virus matrix proteins regulate budding and egress from the host cell membrane. Identification of lipids that promote or inhibit budding as well as differences among the Filovirus family members could provide better means to reducing viral replication and treating infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI081077-09
Application #
9733090
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Dupuy, Lesley Conrad
Project Start
2009-07-01
Project End
2020-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
9
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Purdue University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
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Del Vecchio, Kathryn; Stahelin, Robert V (2018) Investigation of the phosphatidylserine binding properties of the lipid biosensor, Lactadherin C2 (LactC2), in different membrane environments. J Bioenerg Biomembr 50:1-10
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Oda, Shun-Ichiro; Noda, Takeshi; Wijesinghe, Kaveesha J et al. (2016) Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression. J Virol 90:1839-48
Gc, Jeevan B; Johnson, Kristen A; Husby, Monica L et al. (2016) Interdomain salt-bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization. Protein Sci 25:1648-58

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