Shelly, Spencer
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Publication Defining A Regulatory Role For The Hsv Glycoprotein B Membrane Proximal Region In Membrane Association(2013-01-01) Shelly, SpencerHerpes simplex virus (HSV) entry requires four essential glycoproteins (gD, gH/gL, and gB) to enable fusion between the virion envelope and the cellular membrane. The fusion cascade is activated by gD binding to one of its receptors, nectin-1 or HVEM. Glycoprotein B (gB), a class III viral fusion protein, mediates the fusion reaction, while data indicates that gH/gL acts as a regulator of gB. gB is trimeric and has a 773 amino acid ectodomain with a highly hydrophobic membrane proximal region (MPR) (residues 731-773) and two fusion loops (FL) per protomer. The post-fusion structure of gB was solved from the gB(730t) construct, which is truncated to remove the hydrophobic MPR residues. In this dissertation I investigated the MPRs influence on gBs ability to interact with membranes. I hypothesize that the MPR regulates fusion loop exposure by interacting with the fusion loops and masks them until fusion begins. To investigate this process I constructed a series of MPR deletion, truncation, and point mutations using both full-length mammalian expression vectors and purified baculovirus expressed protein. I found that deletions in the MPR from full-length gB resulted in a disruption in cell surface expression in transfected cells. This suggests the MPR is necessary for proper folding or transport of gB. Soluble gB MPR truncations [gB(759t), gB(749t), gB(739t)] were expressed and purified using the baculovirus expression system, and compared to MPR-less gB(730t) and full MPR containing gB(773t). I found that gB containing an MPR segment were all compromised in their ability to bind liposomes in comparison to gB(730t), which lacks any MPR residues. Supporting our hypothesis we found that residues 731 to 739 were sufficient prevent liposome association and mutation of two aromatic residues, F732 and F739, to alanine in gB(739t) restored gBs ability to bind liposomes. Together, my data suggests the MPR does indeed regulate gBs ability to associate with liposomes, and that aromatic residues in the MPR are important for this function. This supports our model that the MPR masks the gB FLs to prevent premature membrane association and adds another layer of regulation to the HSV entry cascade.