Margaret Kielian, Ph.D.

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 such as Semliki Forest virus (SFV) and Sindbis virus (SIN) and flaviviruses such as dengue virus (DV). 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 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 flavivirus and alphavirus membrane fusion proteins 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 SFV fusion protein E1. This structure is strikingly similar to the DV homotrimer. Using the structures as a guide, our lab developed fragments of the SFV and DV 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 are being addressed. We are currently developing in vitro trimers as screens for small molecule inhibitors of virus fusion reactions and lead compounds for new antiviral therapies. Using the in vitro reconstitution system and a novel fluorescent labeling approach, we are addressing the mechanism of fusion protein insertion into the target membrane. Cooperative inter-trimer interactions occur 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. 

During alphavirus biogenesis, the companion protein E2 forms a closely-associated dimer with E1, and protects it from low pH and premature fusion during exocytic transport. During virus entry the mature E2-E1 heterodimer must dissociate at low pH to permit virus fusion. We are studying these key alphavirus pH protection and dissociation steps. The pH protection mechanisms for many viruses are unknown, and we are using rubella virus as a system to define novel mechanisms of pH protection.

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? The available data strongly suggest that the exit pathway of the alphaviruses involves unique cellular proteins. We carried out a genome-wide RNAi screen to identify novel cellular proteins involved in alphavirus entry and exit. We are currently defining the role of these new proteins in the virus lifecycle, and they are exciting new targets for antiviral strategies. We have developed fluorescently tagged viruses to follow alphavirus assembly and budding in real time in infected cells.

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 initial infection, characterization of the role of viral and cellular factors in virus entry and budding, mutagenesis of virus infectious clones to characterize specific steps in fusion, screens for virus fusion and exit inhibitors.