David A. Sanders

Active Mentor - currently hosting PULSe students for laboratory rotations and recruiting PULSe students into the laboratory; serves on preliminary exam committees

Current Research Interests:

Retroviruses, through the process of pseudotyping, can acquire the glycoproteins of certain other enveloped viruses and can utilize them for entry into cells. We have demonstrated, for example, that the Ebola virus glycoprotein can be incorporated into replication-defective retroviruses and that it can mediate viral entry into cells. Our experiments allow us to study the entry of viruses such as the Ebola and Marburg viruses in a fashion that is independent of the other steps in the viral life cycle and to do so in a safe and quantitative manner. Our pseudotyped viruses may also have applications in gene-transfer and gene-therapy experiments. Using the system that we have developed, we have found, for example, that the biochemistry of Ebola virus entry resembles that of bird retroviruses. Our research supports the hypothesis that these viruses shared a common ancestor. This indicates that it is likely that the Ebola virus either currently has a bird as its natural host or that it has evolved from a bird virus.

Our studies have also allowed us to determine the disulfide-bond map of the Ebola glycoprotein and to propose that reduction of the disulfide bond between the two subunits of the Ebola glycoprotein complex, GP1 and GP2, is a critical step in the entry of Ebola virus into cells. Furthermore, we have shown that removal of a region of O-glycosylation of the protein enhances processing and its incorporation into recombinant pseudotyped retroviruses. This modification allows for a greatly improved efficiency of gene transfer by the recombinant viruses. Our recombinant viruses bearing the Ebola virus glycoproteins are particularly suited for gene therapy for diseases such as cystic fibrosis.

We have invented viruses that have the shells of alphaviruses and the cores of retroviruses. These novel pseudotyped viruses have numerous advantages as gene delivery/gene therapy agents. We have recently demonstrated in a collaboration with researchers at the University of Iowa that these viruses have superior capacity for introducing genes into the liver and brain glial cells in vivo. They therefore possess great promise for the treatment of a number of diseases.

We and our collaborators have identified the cysteine residues that participate in the murine leukemia virus envelope-protein intersubunit cystine bridge. Analysis of the sequence surrounding the disulfide-forming residues suggests that a thiol-disulfide exchange reaction plays a critical role in the activation of membrane fusion. Our hypothesis is that thiol-disulfide exchange reactions are fundamental to solving the basic problem of virus structure and entry, i.e., how to be stable outside a cell and yet be able to disassemble during penetration. We have also examined how an organism can evolve to acquire resistance to retrovirus-mediated disease and have shown that this can be achieved through the acquisition of a fusion-defective envelope protein. Our findings suggest an approach to the design of a therapy for the prevention or treatment of AIDS.

In another aspect our research we have determined the three-dimensional structure of the Methanosarcina thermophila acetate kinase in collaboration with the laboratories of Greg Ferry at Penn State and Miriam Hasson at Purdue University. As we had predicted, despite the absence of identity between the sequences of acetate kinases and proteins of previously known three-dimensional structure, the core fold of acetate kinase is the same as that of the glycerol kinase/hexokinase/actin/hsp70 superfamily of phosphotransferases. The superfamily is now known as the ASKHA (Acetate and Sugar Kinases/Hsp70/Actin) phosphotransferases. Biochemical, evolutionary, and geochemical considerations support the proposal that an acetate kinase may be the ancestral phosphotransferase of this very ancient and diverse superfamily. We are continuing our studies investigating the enzymology of acetate kinase and the three-dimensional structures of isobutyrate kinase and exopolyphosphatases, which are also members of the ASKHA phosphotransferase superfamily. Our recent findings provide new insights into the extraordinary processiveness of the bacterial exopolyphosphatases and the mechanism by which bacteria respond to starvation conditions.

Selected Publications:

Buss, K. A., Cooper, D. R., Ingram-Smith, C., Ferry, J.G., Sanders, D. A., and Hasson, M. S. (2001). Urkinase: Structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases. J Bacteriology 183, 680-686.

Sharkey, C. M., North, C. L., Kuhn, R. J., and Sanders, D. A., (2001). Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production. J Virol 75, 2653-2659.

Jeffers, S. A., Sanders, D. A., and Sanchez, A. (2002). Covalent modifications of the Ebola virus glycoprotein. J Virol 76, 12463-12472.

Sanders, D. A. (2003). Ancient viruses in the fight against HIV. Drug Discovery Today 8, 287-291.

Sinn, P. L., Hickey, M. A., Staber, P. D., Dylla, D. E., Jeffers, S. A., Davidson, B. L., Sanders, D. A., and McCray, P. B., Jr. (2003). Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha. J Virol 77, 5902-5910.

Taylor, G. M. and Sanders, D. A. (2003). Structural criteria for regulation of membrane fusion and virion incorporation by the MuLV TM cytoplasmic domain, Virology 312, 295–305.

Kahl, C. A., Marsh, J., Fyffe, J., Sanders, D. A., and Cornetta, K. (2004). Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus. J Virol 78, 1421-1430.

Sanders, D. A. (2004). Ebola virus glycoproteins: guidance devices for targeting gene therapy vectors. Expert Opin Biol Ther 4, 329-336.

Sanders, D. A. and B. L. Wanner (2005). Polyphosphate: From bugs to brains. Nature ELS online.

Strang, B. L., Takeuchi, Y., Relander, T., Richter, J., Bailey, R., Sanders, D. A., Collins, M. K., Ikeda, Y. (2005). Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells. J Virol 79, 1765-1771.

Alvarado, J., Ghosh, A., Janovitz, T., Jauregui, A., Hasson, M.S., and Sanders, D.A. (2006). Origin of Exopolyphosphatase Processivity: Fusion of an ASKHA Phosphotransferase and a Cyclic Nucleotide Phosphodiesterase Homolog. Structure. 14, 1263-1272.

Brindley, M. A., L. Hughes, A. Ruiz, P. B. McCray, Jr., A. Sanchez, D. A. Sanders, and W. Maury (2007). Ebola virus glycoprotein 1: Identification of residues important for binding and postbinding events. J Virol 81, 7702-7709.

Mukhopadhyay, S., Hasson, M.S., and Sanders, D.A. (2008). A continuous assay of acetate kinase activity: Measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate. Bioorg Chem 36, 65-69.