Ebola virus (EBOV) and Lassa virus (LASV) infection is enhanced when cells express phosphatidylserine (PS) binding receptors on the cell surface. PS is a cellular lipid normally restricted to the inner leaflet, or cytoplasmic face, of the cellular plasma membrane (PM). The restriction of PS and other phospholipids to the inner leaflet produces a highly asymmetric membrane in healthy cells. PS is flipped to the outer leaflet of the cellular membrane during calcium signaling and apoptosis, marking them as activated or dying cells, respectively. Cellular enzymes termed flippases and scramblases are responsible for flipping the PS in the membrane. PS is incorporated into the viral membrane during virus budding, when EBOV and LASV particles appropriate a portion of the host cell's plasma membrane as a protective envelope. In order to engage PS receptors, the PS on a viral envelope must be flipped to the outer leaflet.
We aim to elucidate the mechanism by which viral envelopes obtain properly oriented PS, as well as the amount of PS sufficient to interact with PS receptors. We are using a panel of human haploid (HAP1) cell lines lacking PS flippases or PS scramblases to examine the role these proteins play in the replication of EBOV and LASV. We produced vesicular stomatitis virus containing either its native glycoprotein (G), LASV-GP, or EBOV-GP, enabling us to perform experiments under BSL2 conditions. When these recombinant viruses were grown in the HAP1 knock-out cell lines, we identified one flippase and one scramblase that are required for efficient spread of VSV particles containing either the LASV-GP or EBOV-GP, but not VSV-G. This data suggests altering the levels of PS in the outer leaflet of the cellular PM inhibits one or more steps in the viral replication cycle. Virus-like particles resembling either EBOV or LASV were found to contain less surface PS when produced in cells deficient in scramblase activity, also supporting our overall hypothesis that cellular enzymes involved in the production and localization of PS will impact EBOV and LASV entry and spread. We have proposed three specific aims to further examine the role PS plays in viral replication:
Aim 1 : Examine the requirements for cellular scramblase activity in EBOV and LASV entry and virion production;
Aim 2 : Examine the requirements for cellular flippase activity in EBOV and LASV entry;
Aim 3 : Determine the effects of altered cellular PS to EBOV and LASV replication and the viral lipid profile. Using VSV-based pseudoparticles in addition to virus-like EBOV particles and recombinant lymphocytic choriomeningitis virus (rLCMV) containing the LASV-GP, we will determine the role of cellular flippases and scramblases during viral replication, and confirm our results using similar experiments with authentic virus in a BSL4 lab. Experiments will also define the lipidome of these viruses and approaches will focus on quantifying the levels of modified PS on viral particles. Understanding the role PS and other lipids play on LASV and EBOV infectivity will potentially provide new targets for future antivirals.

Public Health Relevance

Phosphatidylserine is a lipid linked to efficient virus entry, yet we do not currently understand the step(s) in the viral replication cycle that require this lipid. This project will examine the role phosphatidylserine plays in entry and budding of hemorrhagic fever viruses, as well as identify additional lipids needed for virus replication. Understanding the interplay between cellular lipids and virus replication may provide new targets for developing antivirals.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI139238-03
Application #
10115579
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Dupuy, Lesley Conrad
Project Start
2019-03-05
Project End
2024-02-29
Budget Start
2021-03-01
Budget End
2022-02-28
Support Year
3
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Georgia
Department
Microbiology/Immun/Virology
Type
Schools of Veterinary Medicine
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602