Human immunodeficiency virus, the etiologic agent of AIDS, is a complex retrovirus of the lentivirus subfamily. The HIV-1 genome contains six accessory gene known as vif, vpr, tat, rev, vpu and nef. The functions of tat and rev are essential for HIV-1 replication, but the functions of the other genes vary depending on the target cells employed for analysis. Contradictory data has been reported for some genes. In the initial funding period the applicant noted in structure-function studies of HIV-1 using a macrophage-tropic molecular clone designated 89.6, that Vpr plays a significant role in the productive infection of primary macrophages as observed for HIV-2 and chimeric HIV-1 containing partial sequences from macrophage-tropic virus. The characteristic features of Vpr noted by several studies including the applicant's are: (i) Vpr is stable in cells, (ii) Vpr has the ability to oligomerize, (iii) Vpr is transported to the nucleus, (iv) Vpr is incorporated into the virus particle, (v) Vpr has a positive effect on HIV-I infection in macrophages, (vi) Vpr prevents establishment of infected cells that chronically produce virus, and (vii) Vpr induces differentiation and arrest cell progression at G2 phase of the cell cycle. Despite the demonstration of these characteristics there is limited information available regarding the essential domains of Vpr for function. The hypothesis to be tested in this proposal is that Vpr has either distinct functional domains contributing to specific features of Vpr or a specific domain contributes to multiple features. It is likely that a combination of these may also be operative. An understanding of the structure-function relationship of Vpr will yield useful information regarding the interrelationship between oligomerization, nuclear localization, virion incorporation and the effect of Vpr at the level of virus infection. Based on preliminary studies and data published by others on vpr, the applicants propose to investigate in detail the following: (I) The requirement of Vpr for incorporation into the virus particle will be determined. The Vpr coding sequences will be altered utilizing several strategies based on secondary structure prediction and molecular modeling studies and characterized using different biochemical biological assays. As Vpr and Vpx incorporated into HIV-1 and -2 Gag directed virus particles respectively, chimeragenesis of Vpr and the related Vpx will be undertaken to define the sequences underlying the specificity of Vpr incorporation into the virus particles; (II) Structural protein Gag p6 domain will be analyzed in detail to localize the residues that participate in the incorporation of Vpr into the virus particle; (III) The molecular mechanism(s) of Vpr incorporation into the virus particle will be elucidated by analyzing the protein-protein interactions involving Gag and Vpr; (IV) The role of Vpr in HIV- I replication will be studied utilizing several Vpr mutants; and (V) The structural constraints associated with Vpr incorporation into the virus particle will be addressed. Vpr- fusion proteins and Vpr linked dimers will be generated and incorporation into virus particles will be addressed. This information will then be used to generate Vpr-fusion proteins that might interfere with HIV-1 replication. The structure- function studies of HIV-1 Vpr in terms of incorporation into the virus particle and its role in viral replication will be useful for understanding AIDS pathogenesis and for development of therapeutic agents.
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