Gary H. Cohen, Ph.D.
Professor, Dept of Microbiology

Microbiology, Virology and Parasitology Program

Address

216 Levy Building
240 S 40 Street
Philadelphia, PA 19104-6002

Office tel.: 215 898-5914
Lab tel.: 215 898-6558
Fax: 215 898-8385
E-mail: cohen@biochem.dental.upenn.edu

Link(s)

Dr. Cohen & Dr. Eisenberg's lab website

Education

Brooklyn College: BS (Biology), 1956.

University of Vermont: PhD (Microbiology), 1964.

University of Pennsylvania: Postdoctoral Fellow (Virology), 1964.

Research Interests

• Herpes simplex virus entry mechanisms; Structure-Function of HSV viral Entry proteins vaccinia virus proteins involved in protection.

Key words: herpes, HSV, virology, glycoproteins, poxvirus, vaccinia, virus entry.

Search PubMed for articles

Description of Research

The research in my laboratory is conducted jointly with Dr. Roselyn J. Eisenberg of the School of Veterinary Medicine , UPenn. Our long term goal is to understand molecular events that mediate virus entry into susceptible cells and promote the pathogenesis of the virus in its human host. We are currently working on two viruses, herpes simples virus (HSV) and vaccinia virus.

Herpesvirus Research

HSV entry requires four envelope glycoproteins, gD, gB and a heterodimer of gH and gL. Binding of gD to its cell receptor triggers virus-cell fusion that requires the concerted actions of gB and gH/gL. Glycoprotein D interacts with either of two protein receptors, called HVEM or nectin-1. HVEM is a member of the TNF receptor superfamily. Nectin-1 is an adhesion molecule in the Ig superfamily located at adherens junctions of cells and neuronal synapses. HSV gD also competes with the immune modulator BTLA for binding to HVEM on T cells. The significance of this for HSV pathogenesis is a goal of our ongoing research. A major accomplishment was to solve the 3-dimensional structure of glycoprotein D bound to HVEM. Additional structural studies showed that the binding sites for both receptors are hidden in the native protein. A major conformational change in gD accompanies receptor binding and also triggers downstream events involving gB and gH/gL. In collaboration with Drs. Katya Heldwein and Steve Harrison (Harvard), we solved the 3-dimensional structure of gB. This glycoprotein has remarkable homology to the receptor binding and fusion protein G of vesicular stomatitis virus (VSV). Two publications describing these exciting results recently appeared in Science. We are using structure based mutagenesis combined with biochemical and immunological approaches to dissect functional regions of gB. In addition we are exploring how gH/gL contributes to HSV induced fusion.

In other studies, we and others have also found that virus entry can occur by at least three different mechanisms: direct fusion with the plasma membrane, pH dependent endocytosis and pH independent but receptor dependent endocytosis. We are now trying to figure out the “rules” that govern which pathway is used in any particular cell type. Finally, we are examining entry using real-time imaging with viruses and/or receptors that bear fluorescent tags.

Poxvirus Research

A major goal of this work is to develop a subunit vaccine against smallpox using proteins from vaccinia virus or from variola virus (smallpox). We cloned several VV envelope proteins in a baculovirus expression system, purified the recombinant proteins and evaluated them for efficacy as vaccines in mouse and monkey models. A combination of three envelope proteins of VV protects mice against a lethal VV challenge. Preliminary studies indicate that this combination is also effective in protecting monkeys against a monkeypox challenge. Studies are underway to test variola homolgoues of the VV proteins in the mouse model of VV infection. A second goal is to use the reagents we develop to ask more fundamental questions about poxvirus entry into susceptible cells. A variety of experimental approaches are being taken, modeled on our herpesvirus work.

Recent Publications

Aldaz-Carroll L, Xiao Y, Whitbeck JC, de Leon MP, Lou H, Kim M, Yu J, Reinherz EL, Isaacs SN, Eisenberg RJ, Cohen GH. Major neutralizing sites on vaccinia virus glycoprotein b5 are exposed differently on variola virus ortholog b6. 2007 Aug; J Virol. 81(15):8131-9.

Jing L, Chong TM, Byrd B, McClurkan CL, Huang J, Story BT, Dunkley KM, Aldaz-Carroll L, Eisenberg RJ, Cohen GH, Kwok WW, Sette A, Koelle DM. Dominance and diversity in the primary human CD4 T cell response to replication-competent vaccinia virus. 2007 May 15 J Immunol.178(10):6374-86.

Cairns TM, Friedman LS, Lou H, Whitbeck JC, Shaner MS, Cohen GH, Eisenberg RJ. N-Terminal Mutants of Herpes Simplex Virus Type 2 gH Are Transported without gL but Require gL for Function. 2007 May; J Virol. 81(10):5102-11.

Hannah BP, Heldwein EE, Bender FC, Cohen GH, Eisenberg RJ. Mutational evidence of internal fusion loops in herpes simplex virus glycoprotein B. 2007 May J Virol.; 81(9):4858-65.

Bender FC, Samanta M, Heldwein EE, de Leon MP, Bilman E, Lou H, Whitbeck JC, Eisenberg RJ, Cohen GH. April, 2007: Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions. 2007 Apr; J Virol. 81(8):3827-41.

Lab

Rotation Projects

1. A number of mutated forms of herpes glycoproteins gD, gB and gH have been created and tested in mammalian expression systems. However, we also want to express these is larger quantities for more careful protein analysis. As a rotation project, the student will subclone the ectodomain of one of these mutant forms into a baculovirus expression system. The student will become skilled in this technology and will learn how to purify the proteins and to analyze their properties. Purified proteins will be used in various biochemical and biological assays, e.g. Westerns, ELISA, BIAcore. The student will learn a variety of molecular biological and virological techniques and will become skilled in the use of the baculovirus expression system as well as protein purification and characterization.
   

2. To clone and express vaccinia virus proteins that are important components of the host immune response using the baculovirus system. Similar strategies and techniques to those described above will be employed. To carry out bioassays, the student will collaborate with others in the lab who are permitted to work with vaccinia virus so that the student is not exposed to the virus.
   

3. Creation of mutations in the genes for one of the herpes or VV glycoproteins under investigation in our lab. Such mutants will enhance our understanding of these proteins and the choice of the particular mutant will depend on the current research in the lab. This project will entail the use of the Quick Change mutagenesis and will allow the student to make and characterize the mutant proteins in a mammalian expression system.
   

4. We recently developed a bimolecular complementation assay to study transient protein-protein interactions that occur among the HSV glycoproteins involved in entry and cell-cell fusion. The assay relies on making fusion proteins with either the N- or C-terminal half of yellow fluorescent protein (YFP) hooked to one end of the protein of interest. If there is an interaction between the two hybrid proteins, it is visualized as a gain of fluorescence by confocal microscopy. We have a state of the art microscope equipped with hardware for doing live-cell confocal studies. All of the necessary expertise for doing these experiments is in place and several people in the lab have projects that involve its use. The student will create one of the chimeric proteins and study its properties by microscopy techniques. The student will acquire knowledge and expertise not only in standard cloning but also in the use of this exciting new visual technology.

Lab personnel:

Eric Lazear, B.S., CAMB graduate student (4th year)
Brian Hannah, B.S., CAMB graduate student (4th year)
Katie Stiles, B.S., CAMB graduate student (3rd year)
Chwang Hong Foo, B.S. CAMB graduate student (3rd year)
Tina Cairns, Ph.D., Research Associate
Florent Bender, Ph.D., Research Associate
Doina Atanasiu, Ph.D., Research Associate
J. Charles Whitbeck, Ph.D., Senior Research Scientist
Huan Lou, BS, Research Specialist
Manuel Ponce de Leon, MS, Research Specialist

Former graduate students:

J.T. Matthews, Ph.D. Director, Aventis Pasteur Group, External Research &. Development, Sanofi Pasteur Inc

C. Seidel-Dugan, Ph.D. Director, New Targets, Oncology Research, Schering-Plough Research Institute, Kennilworth, NJ

D. L. Sodora, Ph.D., Associate Professor, University of Texas

Shan-Ling Hung, Ph.D. May, 1992; Professor of Oral Biology, National Yang-Ming University, Taipei, Taiwan R.O.C.

Deborah Long,, Ph.D. Aug., 1992. Group Leader, Virology program, Wyeth, Inc.

Hsien-Yuan Chiang, Ph.D. Professor of Microbiology, National Defense Medical Center Taipei, Taiwan

Ruth Tal-Singer, Ph.D., Section Chief, Smith-Kline Beecham

Christopher Handler, Ph.D. CRA, Quintiles, Rockville, MD

Anthony Nicola, Ph.D., Assistant Professor, Virginia Commonwealth University

Tao Peng,Ph.D. (Biology Grad. Group) Staff Scientist, Immusol

Sarah Connolly, Ph.D. May 2003. Post-doctoral fellow, Dr. Robert Lamb, Northwestern University.