Margaret Kielian, Ph.D

 

Professor, Department of Cell Biology Chanin Bldg., Room 401 718 430-3638 kielian@aecom.yu.edu Biosketch Laboratory Web Site Complete list of publications

Research interests During infection all enveloped viruses use the essential steps of membrane fusion to enter a cell, and membrane budding to produce infectious progeny viruses. Molecular information on these processes is critical to understanding the infection pathways of enveloped viruses and as a key model for cellular membrane fusion and budding reactions. The research in our laboratory focuses on the molecular mechanisms of virus-membrane fusion and virus budding using the alphavirus Semliki Forest virus (SFV) and the flavivirus dengue virus (DV). The flaviviruses and alphaviruses include many important human pathogens such as dengue, West Nile, and yellow fever viruses. DV is currently of particular concern as it has dramatically reemerged to become endemic in >100 countries, with an estimated 100 million cases of dengue infection per year. There are currently no vaccines or antiviral therapies for DV, and new therapeutic strategies for the flaviviruses and alphaviruses are urgently needed. SFV is a highly developed system to study virus fusion and budding, and is an important experimental model for both alphaviruses and flaviviruses. Both SFV and DV enter cells by endocytic uptake and then fuse their membrane with the endosome membrane in a reaction triggerd by the low pH of the endocytic vesicle. The flavivirus and alphavirus membrane fusion proteins are members of the "class II virus fusion proteins". These structurally related proteins refold during fusion to form a homotrimer that mediates virus fusion and infection. In collaboration with Dr. Félix Rey, we determined the structure of the homotrimer conformation of the SFV fusion protein E1. This structure is strikingly similar to the DV homotrimer. Using the structures as a guide, our lab has recently developed fragments of the SFV and DV fusion proteins that act as dominant-negative inhibitors of SFV and DV fusion and infection. We are using these protein fragments to define the steps of the fusion reaction and also as the basis to develop general screens for inhibitors of class II fusion reactions. Such inhibitors will serve as lead compounds to develop new antiviral therapies. E1-membrane insertion and alphavirus fusion are strikingly dependent on the presence of cholesterol in the cell membrane. The control of E1-membrane insertion and the mechanism of E1's cholesterol and pH-dependence are not understood, and we are using biochemistry, virus genetics and in vitro mutagenesis of SFV and DV infectious clones to address these questions. SFV exits by budding through the plasma membrane of the infected host cell. Little is known about budding of alphaviruses or flaviviruses, although it is clear that budding is highly specific and produces very organized virus particles. How does this happen and what are the roles of cellular and viral components? We have developed a novel biochemical method to assay and characterize the budding of SFV from the plasma membrane. This system can now be used along with mutagenesis and cellular protein analysis to define the SFV budding reaction. Our lab uses a wide variety of approaches including molecular biology, virus genetics, protein biochemistry, cell biology, fluorescence spectroscopy, and structural biology. Potential research projects include: the molecular mechanism of the homotrimer during membrane fusion, screens for DV and SFV fusion inhibitors, mutagenesis of virus infectious clones to characterize specific stages of fusion, the mechanism of E1-membrane insertion and the role of cholesterol, the structure and function of the E1 transmembrane and stem regions, the role of viral and cellular factors in virus budding.

Recent publications Gibbons, D. L., I. Erk, B. Reilly, J. Navaza, M. Kielian*, F. A. Rey, and J. Lepault*. (2003) “Visualization of the target-membrane-inserted-fusion protein of Semliki Forest virus by combined electron microscopy and crystallography.” Cell 114:573-583. *Joint corresponding authors.  Gibbons, D.L., M.-C. Vaney, A. Roussel, A. Vigouroux, B. Reilly, J. Lepault, M. Kielian*, and F. A. Rey*. (2004) “Conformational change and protein-protein interactions of the fusion protein of Semliki Forest virus.” Nature 427, 320–325. *Joint corresponding authors. Chanel-Vos, C. and M. Kielian. (2004) “A conserved histidine in the ij loop of the Semliki Forest virus E1 protein plays an important role in membrane fusion.” J. Virol. 78:13543-13552. (cover article)  Liao, M., and M. Kielian. (2005) “Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus-membrane fusion”. J. Cell Biol. 171:111-120.   Kielian, M., and F.A. Rey. (2006) “Virus membrane fusion proteins: more than one way to make a hairpin.” Nature Rev. Microbiol. 4:67-76. Kielian, M. (2006) “Class II virus –membrane fusion proteins”. Virol. 344:38-47. Chanel-Vos, C., and M. Kielian. (2006) “Second-site revertants of a Semliki Forest virus fusion-block mutation reveal the dynamics of a class II membrane fusion protein.” J. Virol. 80:6115-6122. Liao, M., and M. Kielian. (2006) "Site-directed antibodies against the stem region reveal low pH-induced conformational changes of the Semliki Forest virus fusion protein." J. Virol. 80:9599-9607. Liao, M., and M. Kielian. (2006) "Functions of the stem region of the Semliki Forest virus fusion protein during virus fusion and assembly." J. Virol. 80:11362-11369. [Selected for highlight by editors.] Taylor, G.M., P. I. Hanson, and M. Kielian. (2007). "Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding." J. Virol. 81:13631-13639.

Margaret Kielian: Research interests | Biosketch Faculty research at a glance Birshtein | Bouhassira | Edelmann | Fyodorov | Keogh | Kielian | Kitsis | Nathenson | Query Scharff | Schildkraut | Shafritz | Singer | Skoultchi | Stanley | Steidl |Warner | Ye   Home