Professor, Department of Cell Biology
Samuel H. Golding Chair in Microbiology
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.
Our research focuses on the molecular mechanisms of virus-membrane fusion and virus budding using alphaviruses and the closely related virus Rubella virus, and flaviviruses such as dengue virus (DENV). The flaviviruses and alphaviruses include many important human pathogens such as dengue, West Nile, and Chikungunya viruses, which cause millions of human infections each year. There are currently no vaccines or antiviral therapies for these viruses, and new strategies are urgently needed.
Alphaviruses, Rubella virus and flaviviruses enter cells by endocytic uptake and then fuse their membrane with the endosome membrane in a reaction triggered by the low pH of the endocytic vesicle. The membrane fusion proteins of these viruses are structurally related proteins and refold during fusion to a homotrimer conformation that mediates virus fusion and infection. In collaboration with Dr. Félix Rey, we determined the structure of the homotrimer of the alphavirus fusion protein E1. This structure is strikingly similar to the DENV homotrimer. Using the structures as a guide, our lab developed fragments of the alphavirus and DENV fusion proteins that act as dominant-negative inhibitors of virus fusion and infection. We have reconstituted trimer formation in vitro on target membranes using purified proteins.
Many important questions on the molecular mechanism of membrane fusion remain. We are investigating the mechanism of fusion protein insertion into the target membrane using in vitro reconstitution and fluorescent labeling approaches. Cooperative interactions occur between trimers in vitro but their functional role is controversial and untested. We seek to define these E1 contacts and to determine if they play a critical role for alphaviruses and for other fusion proteins. We are investigating the novel calcium requirement for the insertion of the Rubella virus fusion protein into target membranes.
During alphavirus and flavivirus biogenesis, a companion protein forms a closely-associated dimer with the fusion protein, and protects it from low pH and premature fusion during exocytic transport. This companion protein must then dissociate to permit virus fusion. We are studying these key pH protection and dissociation steps. The pH protection mechanisms for many other viruses are unknown, and we are using Rubella virus as a system to define novel mechanisms of pH protection.
Alphaviruses exit by budding through the plasma membrane of the infected host cell. Little is known about the budding of either alphaviruses or flaviviruses, although it is clear that budding is highly specific (excluding host proteins) and produces very organized virus particles. How does this happen and what are the roles of cellular and viral factors? We have developed fluorescently tagged alphaviruses to follow virus assembly and budding in real time in infected cells. We are investigating how the virus spreads from cell to cell, how the internal RNA-capsid core is recruited to the site of budding, and how the envelope proteins exclude host proteins from the budding site. The cell plasma membrane and cytoskeletal network are extensively remodeled during budding and we are defining the mechanisms and signaling pathways that mediate remodeling. We carried out a genome-wide RNAi screen to identify host factors involved in alphavirus entry and exit, and we are currently defining the role of these novel proteins in the virus lifecycle.
Our lab uses a wide variety of approaches including molecular biology, virus genetics, protein biochemistry, live cell imaging, cell biology, fluorescence spectroscopy, small molecule and RNAi screens, and structural biology. Potential research projects include: use of fluorescently tagged viruses to follow steps in virus assembly and budding, characterization of the role of viral and cellular factors in virus entry and exit, mutagenesis of virus infectious clones to characterize specific steps in fusion and pH protection.
Please see PubMed link on the upper right.
More Information About Dr. Margaret Kielian
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Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
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Bronx, NY 10461