In this application the Principal Investigator proposes to examine the structure-function relationships of two homologous HIV-regulatory proteins, Vpr and Vpx, that are incorporated in virus particles. He and his associates will examine HIV-1/2 Vpr and HIV-2 Vpx. In addition to providing a clearer definition of the mechanism of action of these proteins, the researchers will utilize these sequences to target heterologous proteins into virions which may be useful as an antiviral strategy. They will focus on the following specific questions: (1) What sequences in Gag are required for packaging Vpr and Vpx? (2) What sequences in Vpr and Vpx are required for virion incorporation? (3) Can Vpr and Vpx direct heterologous proteins to virions and define the minimal domains for virion incorporation? (4) What is the function of Vpr and Vpx in retrovirus replication? In answering Question #1 the researchers will use a vaccinia virus expression system to identify Gag determinants required for specific Vpr and Vpx packaging into virus-like particles and chimeric HIV-1/2 Gag particles to identify the determinants necessary for virion incorporation and chimeric RSV/HIV Gag particles to identify determinants sufficient for virion incorporation. Also, they will assess direct interactions of Vpr or Vpx with Gag by co- immunoprecipitation experiments, using GST-fusion proteins or the yeast two-hybrid system. In answering Question #2 the researchers will examine the domains of Vpr which are required for virion incorporation, in particular the C-terminal basic amino acid-rich sequences. They will examine sequences in Vpx required for virion incorporation, with particular focus on the Cys/His-rich domain and the amphipathic helix. In answering Question #3 the researchers will examine whether GST-Vpr or GST-Vpx fusion proteins can be targeted to virions. In addition, they will ask whether Gag-Vpr fusion protein has transdominant inhibitory effects on virus assembly. In answering Question #4 the researchers will determine if Vpx is localized in the nucleus and examine the role of Vpx in virus replication, in particular, to determine if it regulates nuclear DNA import.They will determine which sequences in Vpr and Vpx are required for nuclear import and identify cellular proteins mediating Vpr or Vpx effects using the yeast two-hybrid system.
