Herpes simplex viruses (HSVs) cause a variety of human diseases, from mild cold sores to more serious ones such as encephalitis. This large DNA virus contains 11 glycoproteins in its envelope, of which 4 are essential for virus entry into susceptible cells, called gB, gD, gH and gL. Virus entry and HSV glycoprotein induced cell-cell fusion involve an interaction of gD with a specific cell receptor, followed by fusion of the envelope with a cellular membrane that requires gB and the gH/gL heterodimer. Whereas gD is present only in alphaherpesviruses, gB and gH/gL homologues are found in all herpesviruses. This application focuses on the processes of HSV entry and HSV glycoprotein-induced cell-cell fusion. Because the C-terminus of the gD ectodomain folds back towards the N-terminus of the native protein, receptor binding sites are hidden. Binding of gD to a receptor therefore requires a conformational change to gD that moves the C-terminus of the gD ectodomain away from the core of gD. This movement and the residues of gD that become exposed somehow trigger next steps of virus-cell and cell-cell fusion carried out by gB and gH/gL. These steps are largely unknown. Structural data show that gB is a viral fusogen, but it only functions in concert with gH/gL. Moreover, although the domain structure of gB is now known, the functions of the individual parts of this complex protein remain to be fully elucidated. My laboratory has constructed mutants of gD, gB and gH that are unable to perform their roles in virus-cell or cell-cell fusion, but the reasons for their null phenotypes are not yet understood. We will use a combination of biochemical, physical chemical, mutational and visual approaches to understand the mechanism by which the four HSV glycoproteins cooperate to carry out virus-cell and cell-cell fusion. State of the art techniques such as liposome flotation, FRET based and fluorescent based assays of lipid mixing (hemifusion) and content mixing (full fusion) will be employed to dissect the phenotypes of the various glycoprotein mutants. We will use optical biosensor (BIAcore) Quartz Crystal Microbalance (Q-sense) technology to examine changes to immobilized intact virions exposed to soluble receptors and monoclonal antibodies under various environmental conditions. We will study heterologous interactions of the quartet of glycoproteins that occur before and during cell-cell fusion using a newly developed assay involving bimolecular complementation of yellow fluorescent protein. This novel approach will be coupled with live cell confocal microscopy to visualize interactions that occur during cell-cell fusion in real time.
Two specific aims are proposed: 1) to enhance our understanding of gB structure and function;and 2) to study how the HSV glycoproteins cooperate to accomplish virus-cell and cell-cell fusion. These studies will enhance our understanding of HSV entry, of virus cell fusion in general, and may suggest targets for novel therapeutics.

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 together to allow the virus to enter host cells. My research may suggest 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
Research Project (R01)
Project #
5R01AI076231-15
Application #
8212467
Study Section
Virology - A Study Section (VIRA)
Program Officer
Challberg, Mark D
Project Start
2008-02-01
Project End
2013-08-12
Budget Start
2012-02-01
Budget End
2013-08-12
Support Year
15
Fiscal Year
2012
Total Cost
$370,163
Indirect Cost
$125,138
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Gallagher, John R; Atanasiu, Doina; Saw, Wan Ting et al. (2014) Functional fluorescent protein insertions in herpes simplex virus gB report on gB conformation before and after execution of membrane fusion. PLoS Pathog 10:e1004373
Lazear, Eric; Whitbeck, J Charles; Zuo, Yi et al. (2014) Induction of conformational changes at the N-terminus of herpes simplex virus glycoprotein D upon binding to HVEM and nectin-1. Virology 448:185-95
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
Gallagher, John R; Saw, Wan Ting; Atanasiu, Doina et al. (2013) Displacement of the C terminus of herpes simplex virus gD is sufficient to expose the fusion-activating interfaces on gD. J Virol 87:12656-66
Atanasiu, Doina; Cairns, Tina M; Whitbeck, J Charles et al. (2013) Regulation of herpes simplex virus gB-induced cell-cell fusion by mutant forms of gH/gL in the absence of gD and cellular receptors. MBio 4:
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
Lazear, Eric; Whitbeck, J Charles; Ponce-de-Leon, Manuel et al. (2012) Antibody-induced conformational changes in herpes simplex virus glycoprotein gD reveal new targets for virus neutralization. J Virol 86:1563-76
Bernstein, David I; Earwood, Julie D; Bravo, Fernando J et al. (2011) Effects of herpes simplex virus type 2 glycoprotein vaccines and CLDC adjuvant on genital herpes infection in the guinea pig. Vaccine 29:2071-8
Cairns, Tina M; Whitbeck, J Charles; Lou, Huan et al. (2011) Capturing the herpes simplex virus core fusion complex (gB-gH/gL) in an acidic environment. J Virol 85:6175-84

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