My lab works on enveloped RNA virus assembly and replication. These viruses which are rapidly evolving alongside our immune system include HIV, Ebola and Influenza virus. Providing alternative therapeutic solutions to vaccination requires a full molecular understanding of how these pathogens replicate and hijack cellular machinery.

In this talk I will highlight our work on HIV. HIV incorporates its own protease which upon activation will transform the virus from immature to the infectious mature state. HIV also recruits cellular proteins termed Endosomal Sorting Complexes Required for Transport (ESCRTs) to facilitate its release from the host cell. We have shown that disrupting the balance between budding and protease activation will result in release of non-infectious virus. I will show how we use a variety of single molecule imaging as well as biochemical and virological tools to understand the molecular choreography between ESCRTs and HIV proteins during release of infectious virus.

Iron-sulfur (Fe-S) clusters are common cofactors found in essentially all living organisms. In humans, Fe-S clusters are made predominately in the mitochondria, but assembly machinery is also present in the cytosol. However, when mitochondrial Fe-S cluster assembly is inhibited, some cytosolic Fe-S cluster proteins are not able to convert to their holo forms due to lack of cluster. One hypothesis is that an Fe-S cluster precursor molecule is transported out of the mitochondria, but we believe this molecule to be the Fe-S cluster itself, coordinated to four glutathione molecules [2Fe-2S](GS)₄. The glutathione coordinated cluster has been shown to be stabilized through salt bridges between glutathione molecules. Through liposome transport studies, we have shown that ABC transporter S. cerevisiae Atm1p is able to transport [2Fe-2S](GS)₄ clusters. The ATPase activity of Atm1p is stimulated in the presence of both glutathione and [2Fe-2S](GS)₄. Michalis-Menton kinetics show that [2Fe-2S](GS)₄ lower the K_m for Mg-ATP for WT Atm1p, which may lay insight onto the mechanism of cluster export out of the mitochondria.

Tau is a microtubule associated protein that normally functions as a monomer in league with the microtubule cytoskeleton. However, in Alzheimer's disease (AD), tau aggregates to form filamentous inclusions in cell bodies (neurofibrillary tangles, NFT) and cell processes (dystrophic neurites and neuropil threads). The appearance of tau-bearing lesions correlates with neurodegeneration and cognitive decline, consistent with a connection between tau aggregation and disease progression. Indeed, in biological models, tau aggregates are toxic, with potency inversely proportional to aggregate size. However, the relationship between these species and the aggregation pathway is unknown.

Here we investigate the aggregation mechanism of tau protein in vitro in an effort to identify interactions that manifest size dependence. The fit of a mathematical model describing simple nucleation-elongation polymerization to aggregation time-series, consistently overestimated filament growth rate while underestimating filament length distribution, indicating the presence of a secondary process in the pathway. With the addition of an end-to-end annealing term to the mathematical model in equilibrium with filament fragmentation, we found improved fits for both time series and filament length distributions. Using filament mixing and shearing experiments, we indeed identified end-to-end annealing as a novel secondary interaction of nascent tau filaments. In addition to quantifying the intrinsic rate constants for annealing and fragmentation, the model provided evidence for their dependence on filament length. The results indicate interactions is length dependent. We propose that heterotypic interactions at filament ends are candidate mediators of toxicity in biological models.