Herpes simplex virus causes human disease, ranging from cold sores to more serious infections, some of which are life threatening. Our goal has been to delineate the mechanisms governing HSV entry and HSV-induced cell fusion. In contrast to most viruses, HSV utilizes four glycoproteins in a multi-step process for fusion and entry. Characterization of these events will provide paradigms for virus-mediated fusion in general. The four glycoproteins gB, gD, gH and gL are essential for entry. Crystal structures of gB, gH/gL, gD and gD bound to its receptors have provided critical insights into how these proteins function in virus entry and cell fusion. gD is the receptor-binding protein, while gB and gH/gL form the core fusion machinery for all herpesviruses. The structural homology of gB with G, the fusion protein of VSV, suggests that gB is a fusion protein in all herpesviruses. Here we will study the mechanism used by gB to cause fusion. Unlike G, gB does not function on its own but requires gH/gL. However, gH/gL bears no structural similarities to viral fusion proteins. Moreover, gH/gL and gB can effect fusion when the two proteins are in separate cells (in trans). We postulated that gH/gL acts as a regulator that causes gB to undergo its conformational changes and effect fusion. We postulate that the quartet of HSV glycoproteins act in a sequential fashion. This process begins when gD engages one of its receptors, undergoes conformational changes that allow it to activate the regulatory function of gH/gL. This form of gH/gL then stimulates one or more steps that convert gB from its inert prefusion state to its postfusion form. Although the structure of the gB postfusion form is known, there is no concept of structure for gB or the event that takes place in its conversion. To test our main hypothesis, we propose two specific aims: 1) To learn how gB is converted from a pre to a postfusion state and 2) To examine how fusion involving gD, gB and gH/gL is regulated. We will study the structure of gB and dissect the complex it forms with gH/gL on cells and in vitro using a bead based assay. This high throughput assay will allow us to test the inhibitory effect of peptides and small molecules on the gB/gH/gL interaction. We have identified monoclonal antibodies (Mabs) that distinguish the two forms of gB and distinguish WT from mutant forms of the protein. We also constructed a disulfide-locked mutant that we believe may be a prefusion form of gB, and we have embedded YFP and CFP in gB for possible FRET-based experiments. In collaboration with Dr. A. Steven (NIH) we will visualize WT gB and mutant proteins bound to Fab fragments of gB Mabs. We will use a novel assay that measures the rate of fusion in live cells and use it to study parameters that affect regulation of fusion. Using this assay we have identified hypo- and hyperfusogenic mutants. We will examine the effect of gB Mabs and inhibitory peptides on the rate of fusion to understand more about the mechanisms that regulate the fusion pathway and gB function. Our work has strong clinical significance, as each step in the fusion pathway is a potential target for therapeutics and vaccines against HSV-mediated disease.

Public Health Relevance

Herpes simplex virus (HSV) causes many human diseases but the most common are cold sores and encephalitis. The outer surface of the virus contains the proteins that are needed to gain entry into host cells and cause disease and I use molecular biological approaches to understand how these proteins work to allow the virus to enter host cells. My research may suggest new therapeutic targets that will result in new treatments to stop the virus from gaining entry into its favorite target cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
2R56AI076231-16
Application #
8714298
Study Section
Virology - A Study Section (VIRA)
Program Officer
Challberg, Mark D
Project Start
1997-07-15
Project End
2014-07-31
Budget Start
2013-08-13
Budget End
2014-07-31
Support Year
16
Fiscal Year
2013
Total Cost
$361,900
Indirect Cost
$126,900
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Cairns, Tina M; Huang, Zhen-Yu; Whitbeck, J Charles et al. (2014) Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans. J Virol 88:12612-22
Whitbeck, J Charles; Huang, Zhen-Yu; Cairns, Tina M et al. (2014) Repertoire of epitopes recognized by serum IgG from humans vaccinated with herpes simplex virus 2 glycoprotein D. J Virol 88:7786-95
Atanasiu, Doina; Saw, Wan Ting; Gallagher, John R et al. (2013) Dual split protein-based fusion assay reveals that mutations to herpes simplex virus (HSV) glycoprotein gB alter the kinetics of cell-cell fusion induced by HSV entry glycoproteins. J Virol 87:11332-45
Krummenacher, Claude; Carfi, Andrea; Eisenberg, Roselyn J et al. (2013) Entry of herpesviruses into cells: the enigma variations. Adv Exp Med Biol 790:178-95
Maurer, Ulrike E; Zeev-Ben-Mordehai, Tzviya; Pandurangan, Arun Prasad et al. (2013) The structure of herpesvirus fusion glycoprotein B-bilayer complex reveals the protein-membrane and lateral protein-protein interaction. Structure 21:1396-405