Claude Krummenacher, Ph.D.

Assistant Professor, Department of Biology

Office Address:
215 Levy Building
240 S. 40th St.
Philadelphia PA 19104

Telephone: 215-898-6553
Fax:  215-898-8385

krumm@biochem.dental.upenn.edu

RESEARCH SUMMARY

My studies focus on the nectins, a family of cell adhesion molecules used as cellular receptors by neurotropic herpesviruses. My overall goal is to understand how herpes simplex virus (HSV) uses receptors from the nectin family for entry, spread and pathogenesis.
Toward that goal, three lines of research are being developed:

1. To determine how HSV uses and subverts the normal functions of nectin-1 during viral entry and spread.
2. To define how the cell responds when nectin-1 engages cellular versus viral ligands.
3. To define how binding of nectin-1 to HSV gD triggers virus entry

Members of the nectin family interact with each other at various specialized cell contacts such as synapses in neurons or adherens junctions of the epithelium. For instance, nectin-1 interacts with nectin-3 at synapses and plays a role in ocular and craniofacial development. However, intracellular effects of ligand binding to nectins remain mostly unknown. Nectin-1, and nectin-2 are used by HSV and other alpha-herpesviruses to enter epithelial cells and neurons.
The viral ligand for nectin-1 is the envelope glycoprotein D (gD) and this interaction is a crucial step for HSV entry. At the molecular level, this interaction induces conformational changes in gD that activate the complex mechanism leading to membrane fusion and entry. At the cellular level, gD interferes with the ability of nectin-1 to mediate cell aggregation. This may be the result of direct competition with nectin-1 natural ligands or by inducing an adverse cellular response.

My interest is to understand how nectin-1 mediates cell adhesion and how HSV subverts this natural function during its entry into cells and its spread between cells. Several approaches are being used in the lab.

To study interactions between nectin-1 and its natural and viral ligands at the molecular and structural levels, I use proteins purified from the baculovirus expression system. These proteins are used in various biochemical, virological and cellular assays to define the consequences of nectin-1 binding to gD during entry. This will improve our understanding of molecular requirements for these interactions and lead to structural/conformational determination of these complexes.

Studies of the mechanism of action and regulation of nectins at the cellular level are another facet of my research. First, I am looking at the cellular responses to nectin-1 binding at cell contacts during establishment of adherens junctions. Second I am looking at the signals mediated by nectin-1 during HSV infection. Recent developments in confocal live cell imaging make it possible to look directly at nectins in situ. Various cell lines expressing nectin-1 tagged with GFP, YFP and CFP have been made and allow me to assess the response of nectins upon ligand binding in real time. Fluorescent tagged viruses are being used to study the role of nectin-1 during HSV binding and endocytosis.

Potential rotation projects include:

1. Analysis of the effects of HSV infection on cell junctions. This project involves cell culture, transfections, confocal microscopy and HSV infection, and will give the student training in virology and cell biology.
2. Analysis of the consequences of HSV gD binding to cells expressing nectin-1. This project includes cell culture, transfections and confocal microscopy. Handling purified proteins will also be part of this project involving training in cell biology and protein characterization.
3. Characterization of nectin-1 with its various ligands. This project involves cloning, production and purification of proteins in the baculovirus system. Purified proteins will be used in experiments in vitro (Western blot, ELISA, optical biosensor assays) as well as in cell culture (CELISA, virus entry assays) and will give the student training in molecular biology, virology and protein biochemistry.

Selected Publications:

Krummenacher, C., A.Carfi, R.J. Eisenberg and G.H. Cohen. 2006. Herpesvirus entry into cells: The Enigma Variations. In S. Pöhlmann and G. Simmons, eds. Viral entry into host cells. Landes Bioscience. Vol. in press. Online edition: http://www.eurekah.com/chapter/3035

De Regge, N, H.J. Nauwynck, K. Geenen, C. Krummenacher, G. Cohen, R. Eisenberg, T.C. Mettenleiter and H.W. Favoreel. 2006. Alpha-herpes virus gD interaction with sensory neurons triggers formation of varicosities that serves as virus exit sites. J. Cell Biol. 174:267-275

Krummenacher, C., V.M. Supekar, J.C. Whitbeck, E. Lazear, S.C. Connolly, R.J. Eisenberg, G.H. Cohen, D.C. Wiley and A. Carfi. 2005. Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry. EMBO J. 24:4144- 4153.

Simpson S. A., M. D.Manchak, E.J. Hager, C. Krummenacher, J. C. Whitbeck, M. J. Levin, C. R. Freed, C. L. Wilcox, G. H. Cohen, R. J. Eisenberg and L. I. Pizer. 2005. Nectin-1/HveC mediates herpes simplex type-1 entry into primary human sensory neurons and fibroblasts. J. Neurovir. 11:208-218.

Krummenacher, C., F. Baribaud, M. Ponce de Leon, I. Baribaud, J.C. Whitbeck, R. Xu, G.H. Cohen and R.J. Eisenberg. 2004. Comparative usage of herpesvirus entry mediator A and nectin-1 by laboratory strains and clinical isolates of herpes simplex virus. Virology 332: 286-299.

Krummenacher, C., I. Baribaud, R.J. Eisenberg and G.H. Cohen. 2003. Cellular localization of nectin-1 and glycoprotein D during herpes simplex virus infection. J. Virol. 77: 8985-8999.

Krummenacher, C., I. Baribaud, J.F. Sanzo, G.H. Cohen and R.J. Eisenberg. 2002. Effects of herpes simplex virus on structure and function of nectin-1/HveC. J. Virol. 76: 2424-2433.

RECENT PUBLICATIONS