Herpes Simplex Virus (HSV) infects the majority of people worldwide, causing oral and genital lesions among healthy individuals and life-threatening disseminated disease among the immunocompromised. HSV enters cells by merging its lipid envelope with the plasma membrane of the cell, in a process called membrane fusion. Fusion requires four proteins: the receptor-binding glycoprotein D, a heterodimeric accessory protein composed of glycoproteins H and L, and viral fusion glycoprotein B (gB). According to the current model, gB interacts with membranes via its two fusion loops, FL1 and FL2, and undergoes conformational changes, which pull the cell and viral membranes together during fusion.
In this work, we explored the mechanisms behind several factors that alter gB activity. One such factor is low pH. Depending on the cell type, HSV entry can happen either at the plasma membrane or in endosomes, and the latter usually requires low pH. The role and mechanism of low pH in HSV entry are major questions in the field. We crystallized gB fusion loop mutants in the postfusion conformation at both acidic and basic pH, and showed that low pH causes FL2 to relocate locally. We found that this relocation and other local changes were likely responsible for previously observed pH-dependent antigenic changes in gB. These changes were distinct from the major fusogenic conformational changes observed in other fusion proteins.
The crystallized gB mutants were fusion-null, and each contained a point mutation of a large, hydrophobic side chain in FL1. We found that the mutations did not alter the confirmation of FL1, suggesting that the fusion-null phenotype could be due to the lack of a hydrophobic residue at these positions.
"Rate-of-entry" mutations in gB result in HSV entering cells much more quickly, and correlate with increased pathogenicity. We crystallized two known rate-of-entry mutants in their postfusion conformations and found no differences between their structures and wild-type gB. Additionally, using thermal denaturation, we found the mutants to have wild-type stability. We propose that these mutations do not act by stabilizing the postfusion form but may instead stabilize a transition state of gB to affect the entry rate.
The prefusion conformation of gB has never been characterized, possibly due to its metastable nature. We generated a novel model of gB in its prefusion conformation based on the conformational changes of the structurally similar fusion protein G from Vesicular Stomatitis Virus. Our gB model provides insight into the mechanism of fusion inhibition by several peptide inhibitors and suggests a method of stabilizing the prefusion form of gB by engineering disulfide bonds, providing a starting point for its future characterization.
Together, we characterized the effect of several mutations as well as low pH on postfusion gB, all of which provided important insights into their roles during HSV entry. These observations have improved our current understanding of gB and, coupled with our prefusion model, will continue to do so in the future.
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|Advisor:||Heldwein, Ekaterina E.|
|Commitee:||Bohm, Andrew, Forgac, Michael, Moore, Claire L.|
|School:||Sackler School of Graduate Biomedical Sciences (Tufts University)|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 76/11(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Biochemistry, Virology|
|Keywords:||Entry, Glycoprotein B, Herpes, Membrane fusion, Virus|
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