The Role of Hydrophobic Residues in the Internal Fusion Loop from Ebolavirus GP2 471 views

Ebolavirus, an enveloped Filovirus, causes severe hemorrhagic fever in humans and non-human primates. The viral glycoprotein (GP) is solely responsible for virus-host membrane fusion, but molecular mechanisms of this process remain elusive. Fusion occurs after virions reach an endosomal compartment where GP is proteolytically primed by cathepsins. Fusion by primed GP is governed by an internal fusion loop found in the fusion subunit, GP2. This fusion loop contains a stretch of hydrophobic residues some of which have been shown to be critical for Ebolavirus GP-mediated infection. In this dissertation I present liposome fusion data and the first NMR structures for a complete (54 residue) disulfide-bonded internal fusion loop (Ebov FL) in a membrane mimetic. The Ebov FL induced rapid fusion of liposomes at pH values ≤ 5.5. Consistently, circular dichroism experiments indicated that the α-helical content of Ebov FL in the presence of lipid-mimetics increases in samples exposed to pH ≤ 5.5. NMR structures in dodecylphosphocholine micelles at pH 7.0 and 5.5 revealed a conformational change from a relatively flat extended loop structure at pH 7.0 to a structure with an ~90° bend at pH 5.5. Induction of the bend at low pH reorients and compacts the hydrophobic patch at the tip of the fusion loop forming a fist-like structure. Further analysis of the pH 5.5 NMR structure showed that residues L529, F535, and I544 all point inwards forming a hydrophobic scaffold which supports the fist structure of the fusion loop. These and additional hydrophobic residues in the fusion loop were mutated and screened for liposome fusion activity. Mutations at L529, I544, and the double mutant L529A/I544A were of particular interest and further characterized. The L529A/I544A double mutant was completely inhibited in both lipid mixing and in cell entry of virus-like particles bearing these mutations in full-length GP. The L529A/I544A NMR structure showed significant disruption in the arrangement of hydrophobic residues resulting in inhibited membrane binding and insertion. We show that the consolidation of hydrophobic residues is imperative for membrane insertion and orientation, shedding light on the Ebolavirus fusion process and perhaps, other viral fusion proteins equipped with fusion loops.

PHD (Doctor of Philosophy)

Ebolavirus; NMR solution structure; membrane protein; virus entry; lipid-protein interaction

English

All rights reserved (no additional license for public reuse)

2013-05-22