Gary H Cohen

faculty photo
Professor of Microbiology
Department: Microbiology

Contact information
212 Levy Building
240 S 40th Street.
Philadelphia, PA 19104-6002
Office: (215) 898-5914
Fax: (215) 898-8385
Lab: (215) 898-6553
Graduate Group Affiliations
Education:
B.S. (Biology)
Brooklyn College, 1956.
Ph.D. (Microbiology)
University of Vermont, 1964.
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Description of Research Expertise

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, virus entry.

Description of Research
The research in my laboratory is conducted in collaboration with Dr. Roselyn J. Eisenberg of the School of Veterinary Medicine, UPenn. Our longterm goal is to understand molecular events that mediate virus entry into susceptible cells and promote the pathogenesis of the virus in its human host.

Research on herpes simplex virus (HSV):
Four viral glycoproteins, gB, gD, gH and gL are essential for virus entry and spread. HSV entry is mediated by one of several different receptors. including HVEM (HveA) a TNF receptor, and nectin-1, a cell adhesion molecule that is a member of the Ig superfamily. We showed that purified gD interacts directly with purified HVEM and nectin-1 using such techniques as co-precipitation, ELISA and biosensor. A major accomplishment was to solve the three-dimensional structure of gD with HVEM. The structure of the complex enabled us to carry out structure-based mutagenesis of both proteins. gD mutants with altered receptor usage are being tested in mouse models of HSV pathogenesis. Our future direction is to understand entry events that are downstream of the gD/receptor interaction and involve gB, gH/gL and possibly other cell proteins. Our goal is to carry out structure-function studies similar to those for gD, including solution of their 3-D structures. gB has been crystallized and its structure will soon be known. We recently found that gB partitions into lipid rafts at the time of virus attachment, implicating a raft-specific cellular molecule in entry. In addition, the conformation of gB changes when virions bind nectin-1 at temperatures permissive for entry. Thus, the gD/receptor interaction triggers the downstream events that lead to fusion. This is an active area of ongoing research. In addition, we found that after infection, gD alters the distribution of nectin-1 in cells and also dissociates nectin-1 from its intracellular ligand afadin. Moreover, others and we found that the virus can use both direct fusion and at least two different endocytic pathways of entry, leading us into interesting areas of cell biology.

Poxvirus Research:
We are using similar techniques to those above to study the envelope proteins of vaccinia virus (VV). Live VV is currently the only vaccine available for protection against smallpox (and now monkeypox), a “class A” biological agent according to the CDC. Poxviruses are large complex viruses that are wrapped in two distinct viral envelopes. The inner envelope, called IMV contains at least 5 virion glycoproteins. The outer envelope (called EEV) contains an additional 5 or 6 proteins. Some of these stimulate neutralizing and/or protective antibody responses in animals. Our first goal has been to develop an antibody cocktail that will serve as a replacement for vaccinia immune globulin (VIG), currently offered to smallpox vaccinees with adverse reactions to the vaccine. The second goal is to develop a subunit vaccine against smallpox using VV proteins. A third goal is to understand more about the structure and function of the envelope glycoproteins of this complex virus. Our approach is to express VV proteins in a baculovirus expression system and to purify the recombinant proteins (5 so far) on a scale that is sufficient to develop immunologic reagents (goal 1) and to test the proteins using in-vivo models for efficacy (goal 2). The proteins and antibodies have also been used to address the third goal. The proteins have been evaluated alone and in combination in both mouse and monkey models in a collaboration with investigators at the NIH. A combination vaccine protected mice against a lethal VV challenge and ameliorated the effects of monkeypox in monkeys. These reagents will be further tested against the smallpox virus. Panels of monoclonal antibodies have been developed and are being studied to gain clues about the function of the VV glycoproteins and to test for efficacy in mouse challenge models.

Rotation Projects
1. The mouse homologues of the human HSV receptors HVEM and nectin-1 have been cloned into mammalian expression vectors. These will be useful for carrying out some biological assays. As a rotation project, the student will subclone the ectodomain of one or both of these receptors 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 receptors will be used in binding assays (Westerns, ELISA, biacore) and as immunogens to prepare antisera (the latter done commercially). Understanding the characteristics of these proteins as HSV receptors is critical for understanding the phenotypes of virus mutants carrying mutations in gD that alter receptor tropism. 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 glycoprotein gB or gH/gL that will enhance our understanding of these proteins. This project will entail the use of the Quick Change mutagenesis and will focus on making key mutations that we help the student select, and characterization of the mutant protein using mammalian expression.

Lab personnel:
Eric Lazear, B.S., CAMB graduate student (3rd year)
Brian Hannah, B.S., CAMB graduate student (3rd year)
Katie Stiles, B.S., CAMB graduate student (2nd year)
Chwang Hong Foo, B.S. CAMB graduate student (2nd year)
Claude Krummenacher, Ph.D., Research Associate
Tina Cairns, Ph.D., Postdoctoral Scientist
Florent Bender, Ph.D., Postdoctoral Scientist
J. Charles Whitbeck, Ph.D., Senior Research Scientist
Yi Zuo, DMD, Research Scientist
Huan Lou, BS, Research Specialist
Manuel Ponce de Leon, MS, Research Specialist

Former graduate students:
J.T. Matthews, Ph.D. Senior Scientist, Aventis Pharmaceuticals

C. Seidel-Dugan, Ph.D. Senior Scientist, Elixis Pharmaceuticals

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., Senior Scientist, Smith-Kline Beecham

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

Anthony Nicola, Ph.D., Post-doctoral fellow, Dr. S. Straus, NIH

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

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

Description of Itmat Expertise

Herpes simplex virus samllpox virus, vaccines, mechanism of fusion, glycoproteins

Selected Publications

Richart, S.M., S.A. Simpson, C. Krummenacher, J.C. Whitbeck, L.I. Pizer, G.H. Cohen, R.J. Eisenberg and C.L. Wilcox: Entry of herpes simplex virus into primary sensory neurons in vitro is mediated by nectin-1/HveC. J. Virol. 77: 3307-11, 2003.

Cairns, T.M., R.S.B. Milne, M. Ponce de Leon, D.K. Tobin, G.H. Cohen, and R.J. Eisenberg: Structure-function analysis of herpes simplex virus (HSV-1) gD and gH/gL: clues from gD/gH chimeras. J. Virol. 77: 6731-42, 2003.

Connolly, S.A., D.J. Landsburg, A. Carfi, D.C. Wiley, G.H. Cohen and R.J. Eisenberg: Structure-based mutagenesis of herpes simplex virus glycoprotein D defines three critical regions at the gD/HveA interface. J. Virol. 77: 8127-40, 2003.

Krummenacher C., I. Baribaud, R.J. Eisenberg, and G.H. Cohen: Cellular localization of nectin-1 and glycoprotein D during herpes simplex virus infection. J. Virol. 2003.

Milne, R.S.B., S.L. Hanna, A.H. Rux, S.H. Willis, G.H. Cohen and R.J. Eisenberg: Function of herpes simplex virus type 1 gD mutants with different receptor-binding affinities in virus entry and fusion. J. Virol 2003.

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Last updated: 05/21/2014
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