The small molecular-weight G-proteins are known to be activated in a variety of cell types upon exposure to a range of viruses. However, it is unclear if small G-protein activation during viral exposure is a byproduct of receptor attachment, is important for viral replication, or is playing a role in mediating the inflammatory response. Human rhinovirus (HRV) is unique in that nearly genetically identical viruses bind the ICAM-1 or LDL receptors. Furthermore, HRV is capable of eliciting a signaling response in both epithelial cells, where productive HRV infection occurs, and in macrophages, where there is an immune response. In this study, we will investigate the activation kinetics of Rac, Cdc42, and H-Ras in epithelial cell lines. We will also determine whether Rac is differentially activated when major- and minor-group HRV bind to macrophage ICAM-1 or LDL receptors. Inhibition of Rac in macrophages may attenuate the activation of the stress kinase p38 and the release of the proinflammatory cytokines MCP-1 and RANTES or hinder viral replication. Thus, this project will determine the importance of the G-proteins in establishing the inflammatory microenvironment that is initiated in the human airway upon exposure to rhinovirus.

The small molecular-weight G-proteins are known to be activated in a variety of cell types upon exposure to a range of viruses. However, it is unclear if small G-protein activation during viral exposure is a byproduct of receptor attachment, is important for viral replication, or is playing a role in mediating the inflammatory response. Human rhinovirus (HRV) is unique in that nearly genetically identical viruses bind the ICAM-1 or LDL receptors. Furthermore, HRV is capable of eliciting a signaling response in both epithelial cells, where productive HRV infection occurs, and in macrophages, where there is an immune response. In this study, we will investigate the activation kinetics of Rac, Cdc42, and H-Ras in epithelial cell lines. We will also determine whether Rac is differentially activated when major- and minor-group HRV bind to macrophage ICAM-1 or LDL receptors. Inhibition of Rac in macrophages may attenuate the activation of the stress kinase p38 and the release of the proinflammatory cytokines MCP-1 and RANTES or hinder viral replication. Thus, this project will determine the importance of the G-proteins in establishing the inflammatory microenvironment that is initiated in the human airway upon exposure to rhinovirus.

We are studying microtubules, which are a part of the cytoskeleton and which play a role in cell division, cell mechanics, and intracellular transport. Dynamic studies of microtubule function make use of various fluorescent labels, but these currently available labels suffer form drawbacks such as transient labeling, occlusion of functional sites on the microtubules surface, of structural non-specificity. Our project is to develop and characterize a new, complementary fluorescent labeling technique that avoids these drawbacks.

This new labeling technique uses a fluorescently modified GTP analogue. In cells, microtubules naturally self-assemble from tubulin dimers and GTP. In a test tube, we replicate this process, but add a small amount of fluorescent GTP analogue. The result is microtubules with fluorescent GTP analogue bound to the exchangeable site (E-site) on the tubulin dimer.

Using this technique, we have successfully viewed microtubules. Current work is to further characterize the labeling technique. I am designing experiments to determine where and how tightly the fluorescent GTP analogue binds to the tubulin dimer. Because this technique places a bright fluorophore at a defined location within the microtubule lattice, it may facilitate observations of microtubule dynamics with increased precision.