The Human Immunodeficiency Virus (HIV) is the causative agent of acquired immunodeficiency syndrome ( AIDS). Viral assembly and post-assembly events required for gene activation in the target cell are our major research interests. HIV synthesizes its structural and enzymatic proteins as large polyprotein precursors (the group specific antigen or Gag protein). Proteolytic processing of Gag to generate mature enzymes and structural proteins is essential to the production of infectious virus. The virally-encoded proteinase (PR) is an enzyme which is vitally important for polyprotein processing in the life cycle of the virus. This enzyme, and the Gag polyprotein it cleaves are thus targets of considerable pharmaceutical interest in the search for effective therapies. In fact, drugs that target the proteolytic processing of Gag are part of highly successful anti-retroviral treatment paradigms in current clinical practice. A major focus of our current research is the study of Gag trafficking and assembly. This gene product is sufficient for the assembly and release of virus-like particles (VLPs) and serves as a model for formation of the authentic particle in our studies. A second focus is retroviral capsid structure, assembly, and disassembly. The potential of capsid assembly and disassembly to serve as a target or rational drug design is not yet realized because definition of these processes in molecular detail is just beginning.
Our current research efforts include
1. Role of cellular proteins in retroviral protein assembly: Using a genetic assay for protein-protein interactions, libraries are being examined for cellular proteins that interact with viral structural proteins. The role of one such protein, Tsg101, in trafficking and release of the HIV-1 Gag precursor protein is being examined. Additional cellular proteins involved in the replication of other members of the Retroviridae family also are under study.
2. Structure and dynamics of retroviral proteins: Recombinant Gag, matrix (MA), capsid(CA), and nucleocapsid (NC) proteins of several retroviruses have been cloned, expressed, and purified to near homogeneity by non-disruptive protocols. These are being used for X-ray crystallography and NMR. These studies should complement our structure/function analysis of the assembly and uncoating processes.
3. Identification of determinants of capsid protein assembly and disassembly: Our laboratory was the first to demonstrate that determinants of mature core formation resides within the CA sequence itself through identification of conditions. The morphology of these structures is similar to that of rod-shaped cores produced in natural infection. Specific endopeptidases, scanning transmission electron microscopy, dynamic light scattering, and genetic mapping are being used to identify regions of CA protein involved in subunit interactions. Antiviral drugs that disrupt such interactions may target virus assembly or activation of gene expression in the next target cell.
4. Gag precursor assembly on model membrane systems: Association of the HIV gag polyprotein precursor with cellular membranes is necessary for virion assembly. Following Gag cleavage, dissociation of the mature MA product from the membrane may be required for some post-assembly functions such as fusion with the target cell or subcellular transport of viral components. Fluorescence spectral methods ( in collaboration with S. Scarlata, U.S.B.), genetic, and biochemical techniques are used to determine requirements for membrane- dependent Gag binding and for MA release. The goal of these studies is identification of critical membrane-associated events feasible for targeting of antiviral inhibitors.
5. Regulation of PR activity in its precursor form: How is PR activated to initiate protein processing and what controls, if any, exist to ensure that activation will occur at a specific time in the replication cycle? Precursor forms of the enzyme which cannot autoprocess have been constructed. The specificity and kinetic propertiesare being compared to those of the mature enzyme. Dimerization capability is being assessed using a genetic assay for protein-protein interactions.
6. Molecular basis of resistance to drugs that target PR: Soon after they are administered, variants that are resistant to PRI (PR inhibitors) can emerge as the dominant form of the virus in the host. This seriously reduces treatment efficacy and makes determination of mechanisms underlying resistance to PR inhibitors imperative. Some of the mutations that arise in drug-resistant variants have been subcloned into expression constructs and are under study.