Investigating the effects of pH on alphaviral E3-E2 glycoprotein association, organization, and cellular tropism

<p>In alphaviruses the role of E3 is required in protecting the fusion peptide region of E1 during intracellular transport. Throughout viral processing, the association of E2 and E3 is required for the successful trafficking and incorporation of E1 into the mature virion. This E3-E2 association has been observed to extend to mature virions in the solved structure for the envelope of Semliki Forest virus (SFV) and supported by the solved structure for the entire Venezuelan equine encephalitis virion (VEEV) with exclusive contacts being made between E3-E2. Immunization with monoclonal antibodies against VEEV E3 provided protection for mice challenged by lethal doses of VEEV and suggests potential new targets for antibody neutralization, but it is currently unclear if E3 is retained on mature VEEV virus. Using non-replicating expression systems that avoid virus-culturing artifacts, we discovered that Moloney murine leukemia virus or baculovirus pseudotyped with the alphavirus envelope spike complex of VEEV demonstrates a pH-dependent retention of E3 on mature virus for both mammalian cells and insect cells through indirect-immunofluorescence assays and neutralization studies using polyclonal antibodies against VEEV E3.</p> <p>In studies investigating the impacts of retaining the E3 glycoprotein on mature virus outside of a host cell, we found this retention of E3 decreases receptor-mediated entry of cell targets that can be rescued on cells containing heparan sulfate suggesting viruses containing E3 on mature envelopes can utilize the E3 protein as an attachment factor. We utilized a method for downregulating the cell-surface expression of the natural resistance-associated macrophage protein (NRAMP2), a definitive receptor for the prototypic alphavirus Sindbis (SIND), on cellular targets. We observed a significant decrease in entry of VEEV versus control Ross River (RRV) virus that does not utilize NRAMP2. This inhibition can be rescued with binding to heparan sulfate by VEEV retaining E3. Together, these data suggest that the E3 glycoprotein protects the fusion region of E1 on budded, mature virus dependent on the pH of the extracellular environment. This association potentially serves a role as an attachment factor on virus and presents new binding sites for protein interactions and potential inhibition.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biological Science

Year

2016

Author dc:creator

• Sequra, Jason Michael

Contributors dc:contributor

• Jason Lanman
• Elizabeth J. Taparowsky
• Harm Hogenesch
• David A. Sanders

• Biological sciences
• Alphavirus
• Heparan sulfate
• Membrane fusion
• Venezuelan equine encephalitis virus
• Viral entry
• Viral glycoprotein
• Cell Biology
• Molecular Biology
• Virology

Repository record dc:identifier

https://docs.lib.purdue.edu/open_access_dissertations/997

OAI identifier oai:identifier

oai:docs.lib.purdue.edu:open_access_dissertations-2212