Elucidating the role of ebola virus GP in virus entry and characterization of antibodies against VEE E3

Laura J Hughes, Purdue University

Abstract

Ebola virus causes severe hemorrhagic fever, is a level 4 pathogen that has no treatment, vaccine, or cure, and is classified as a potential biological weapon. Ebola virus enters the host cell via receptor-mediated endocytosis. Cathepsin B and L cleave the glycoprotein (GP) and an undefined low pH event initiates fusion between the viral membrane and the vesicle membrane releasing the nucleocapsid into the cytoplasm. Identifying the receptor and determining the steps in entry would aid in potential drug or vaccine design. Towards this goal, we targeted individual conserved residues within GP for mutational analysis and identified residues involved with receptor binding and post-binding events. To test potential receptors, we attempted to identify a cell line not permissive for entry. Insects have been tested previously for infection permissivity, and insect cells were also believed to not be susceptible for entry. A novel pseudotype virus was created and two insect cell lines were tested for entry. Ebola GP mediated entry into the moth cell line, Sf9 cells. Venezuelan Equine Encephalitis virus (VEEV) is a mosquito-borne positivestrand RNA virus that can cause encephalitis. It enters the cell by receptormediated endocytosis when E2 binds the host cell receptor. E1 initiates fusion of the viral membrane with the vesicle. The precursor protein, pE2, is cleaved to produce E2 and E3. There is strong evidence that supports a role for E3 in inhibiting premature fusion mediated by the alphavirus glycoprotein complex. However, the exact function of E3 is still not well understood. To this end, we expressed and purified soluble VEEV E3 protein that was used to produce antibodies that recognize both E3 and pE2.

Degree

Ph.D.

Advisors

Sanders, Purdue University.

Subject Area

Virology|Immunology

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server
.

Share

COinS