Although much has been learned regarding the genetic structure and molecular biology of HIV-1, much less is understood regarding the nature of the interactions between the virus and the host cell, which leads to the death of CD4+ cells and the ultimate development of fatal immunodeficiency. We have investigated a unique vpr gene present in the primate lentivirus family which includes HIV-1, HIV-2, and the various SIV virus strains. The most noteworthy phenotype of Vpr is the induction of cell cycle arrest specifically at the G2 phase of the cell cycle. This effect of Vpr gives rise to the hypothesis that Vpr may contribute to the induction of immune deficiency by HIV-1 through causing the arrest of infected CD4+ T-cells and preventing their clonal expansion. The development of therapeutics against Vpr as a target may be effective, not only as an anti-viral but also in reversing the immunologic disregulation which may occur as a result of Vpr function. It is critical to develop a better understanding of the domains of Vpr and the functions of those domains which participate in adversely affecting various cellular functions. We propose to utilize genetic analysis of Vpr together with biochemical and structural studies of the interactions between Vpr and a cellular interacting protein HHR23A to develop a more comprehensive picture of the structure-function relationships of this interaction and the phenotypic effects of Vpr on host cell function.
The Specific Aims are: 1. To map the domains Vpr which are involved in binding to the cellular interacting protein HHR23A by genetic and biochemical approaches and to correlate these results with the known functions of Vpr which include a) the induction of G2 arrest, b) nuclear localization c) virion incorporation, d) apoptosis, e) transactivation and f) oligomerization. We expect to develop a comprehensive picture of the regions involved in the interaction between Vpr and the cellular protein HHR23A and the role of these interactions in the differing properties of Vpr. 2. To determine the domains of HHR23A which bind to Vpr. Similar studies to Aim 1 will be conducted except that mutations will introduced into HHR23A, followed by genetic and biochemical assays for interaction with Vpr. 3. To determine the three-dimensional structures of the domains of HHR23A that interact with Vpr. Initially, a modular approach to the structure determination will be taken. Specifically, we will determine the structures of the UBA(1) and UBA (2) domains and the linker region. If the linker region is structured, the UBA(1)-linker UBA(2) structure will also be determined. 4. To identify the specific interactions between HHR23A and Vpr. Structures of mutant HHR23A domains will be investigated on the basis of results obtained on Specific Aim 2. Complexes of the HHR23A UBA(2) domain and Vpr will be studied to determine the changes in the UBA domain on binding Vpr. To determine the three-dimensional structure of Vpr, by itself and/or in complex with UBA(2). Ideally, structures of both the free Vpr and the complex will be determined. However, since Vpr is known to oligomerize, it may only be possible to determine its structure in the complex.
