Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
Gary H. Cohen
Roselyn J. Eisenberg
Herpes simplex virus (HSV) entry into host cells requires the function of the viral envelope glycoprotein (g) B in concert with three other envelope proteins, namely gD and the complex of gH/gL. Glycoprotein D functions in receptor binding and communicates an activation signal to gB and/or to gH/gL to carry out fusion between the viral envelope and cell membranes to release the capsid into the cell and begin the replication process.
In this dissertation, I present a detailed mutagenic analysis of the putative HSV gB fusion loops. I mutated each of the fourteen amino acids that comprise the two loops. In a space filling model of the gB structure, it is apparent that the loops form a single structure, with a hydrophobic ridge at its center. This ridge is comprised of several aromatic and hydrophobic amino acids and is surrounded almost exclusively by charged residues. First, I mutated many of the residues that comprise the hydrophobic ridge. I discovered that five hydrophobic amino acids, four from one loop and one from the other are critical for gB function as mutations to any one of them impair fusion. Four of these comprise the hydrophobic ridge. To my surprise, I also discovered that two charged residues that lie outside the hydrophobic ridge are also important for gB function. Their positions suggest they may support insertion of the hydrophobic ridge into target membranes. These results support my hypothesis that the two loops are an important functional domain that operate cooperatively as one structure.
I expressed several of the point mutants as soluble recombinant gB proteins, and characterized them in several functional assays. I found that mutations of aromatic amino acids to charged ones or charged amino acids to alanines abolished the ability of gB to associate with model liposome membranes. This provides good evidence that these loops are fusion loops that function by inserting into a target membrane. Also, these results suggest that aromatic and charged residues play important roles in target membrane insertion. Other studies have shown that soluble gB can block virus entry, evidence that it interacts with its own entry receptor. A recent report identified a cell surface protein, PILRα, that may fulfill this role. I found that the mutant forms of gB blocked virus entry of virus as efficiently as WT gB, suggesting that the receptor binding region of gB is distinct from the fusion loops. In contrast, these same mutants were significantly impaired in their ability to bind to cells. I suggest that this apparent anomaly may be explained by the possibility that a proportion of gB binds to a surface lipid via the fusion loops. Recently, our lab showed gB and gH/gL interact when triggered for fusion by the addition of soluble gD. In collaboration with another lab member, we show that mutations to the gB fusion loops abrogate gB association with gH/gL.
In summary, my results support the concept that gB functions as a fusion protein and that its two fusion loops act in much the same way as do the fusion loops of VSV G or the class II fusion proteins. Five hydrophobic and two charged residues are most important in the function of the gB fusion loops. I propose that loss of target membrane insertion, impairment of cell binding, and abrogation of gB-gH/gL interaction are all possible mechanisms by which mutations to the gB fusion loops inhibit gB dependent fusion. The remaining enigma is why gB cannot carry out fusion on its own but must act in concert with gH/gL. Future studies may provide the answer to that question. (Abstract shortened by UMI.)
Hannah, Brian, "HSV Glycoprotein B Fusion Loop Architecture and Mechanisms of Fusion Inhibition" (2008). Publicly Accessible Penn Dissertations. 2150.