Novel Approaches to Produce Active, Folded HIV-1 Tat

Tom Alber, University of California, Berkeley
Basic Biomedical Sciences
Innovative, Developmental, Exploratory Award (IDEA)

HIV replication in a host cell requires efficient copying of the viral RNA. This process requires an HIV protein called Tat, but the role of this protein is not very well understood because it has not been possible to produce samples of Tat using standard methods. Tat is especially prone to damage during production and purification, and no strategies currently exist to introduce chemical modifications that are essential for full Tat function. As a result, the samples of Tat that have been made to date are heterogeneous mixtures, partially damaged and missing chemical groups that are normally present in Tat in infected cells. To overcome these problems, we propose to create brand new methods to make Tat. In particular, in order to avoid damage and introduce the proper modifications, we have made Tat in a test tube rather than a cell. Tat is generally considered too big to make by chemical synthesis, but we utilized optimized methods we developed previously to make peptides nearly twice as big. To force Tat to adopt the correct three-dimensional shape, we will invent gentle procedures to allow it to sample many different arrangements and to trap the correct structures by binding them to human partner proteins. The human partner called cyclin T1, for example, will be used as a mold to encourage Tat to adopt the correct shape. These approaches have not been tried before. Our strategies are made possible by unique advances in peptide synthesis methods, recent discoveries about chemical modifications of Tat and new knowledge about the partners of Tat in human cells. This work embodies new ways of thinking about the problems that have limited progress on Tat biochemistry for the last 25 years. The success of this project will enable many studies of Tat that have languished because they are currently considered intractable. By opening up new studies of Tat structure and function, this IDEA award has the potential to deliver new insights about how Tat does its job and how to block these functions with new therapeutics