The Membrane Fusion Mechanism of Herpes Simplex Virus Glycoprotein B.
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... read moreD, 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
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Biochemistry.
Advisor: Ekaterina Heldwein.
Committee: Andrew Bohm, Michael Forgac, and Claire Moore.
Keywords: Biochemistry, Microbiology, and Virology.read less