The human immunodeficiency virus (HIV) is the etiologic agent of AIDS. It is estimated that at least 1.5 million individuals are presently infected with HIV in North America alone and most are expected to develop AIDS. Because there currently is no completely effective drug for treating AIDS, and because no vaccine exists to prevent HIV infection, AIDS clearly remains a major health problem. HIV is a complex retrovirus that encodes several regulatory proteins, including a transcriptional activator. Tat, that is essential for viral replication. Inhibitors of Tat would be expected to block HIV replication or prevent induction of the virus from a latent state, and development of Tat inhibitors is a major objective of this proposal. The process of drug design is expected to be aided significantly by detailed knowledge of the structure of Tat and its RNA- binding site, TAR, and the basic mechanism of transcriptional activation. The interaction of Tat and TAR is required for Tat function, and the amino acids in Tat and nucleotides in TAR responsible for sequence-specific binding have been defined. Preliminary structures of TAR, and of TAR with arginine bound at the Tat-binding site, have been determined by NMR spectroscopy. In this proposal, structural determinants in TAR needed for Tat binding will be defined further by examining the properties of TAR RNA/DNA hybrids, focusing particularly on the backbone conformation required for binding. A new approach to crystallizing TAR is proposed in which TAR is integrated into the structure of a tRNA molecule. Attempts will be made to isolate Tat in its native folded state from mammalian cells for protein structural studies. A related Tat protein from bovine immunodeficiency virus (BIV) has been found to bind to an RNA site similar to HIV TAR at the sequence level, but the structural characteristics of the interaction are rather different. Experiments are described to compare the structures of BIV and HIV Tat-TAR complexes by mapping BIV Tat amino acid contacts by mutagenesis, mapping BIVTAR contacts by RNA chemical modification interference and mutagenesis, and determining the structure of a BIV peptide-TAR complex by NMR. The mechanism of HIV Tat activation, thought to operate at the level of transcriptional elongation, will be examined in the context of an HIV-BIV Tat hybrid protein in which the HIV activation domain is delivered to the HIV promoter without the need for additional cellular TAR-binding proteins. The ability of the hybrid protein to activate transcription will be studied using intact HIV engineered with a BIV TAR site, and by in vitro transcription assays. In the viral experiments, RNase mapping, Northern analysis, nuclear run-on, and Gag protein assays will be used to examine the mechanism of transactivation. A simplified system to study antitermination by Tat will be developed by placing TAR and terminator elements at discrete positions from a strong, nonviral promoter. Attempts will be made to develop transdominant Tat inhibitors based on interfering with the Tat-RAR interaction or interactions of the Tat activation domain with cellular proteins. Small molecule guanidinium analogs that might be expected to bind to TAR will be tested as possible Tat inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
7R01AI029135-08
Application #
2064834
Study Section
AIDS and Related Research Study Section 3 (ARRC)
Project Start
1989-09-30
Project End
1999-04-30
Budget Start
1995-08-01
Budget End
1996-04-30
Support Year
8
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
D'Orso, Iván; Jang, Gwendolyn M; Pastuszak, Alexander W et al. (2012) Transition step during assembly of HIV Tat:P-TEFb transcription complexes and transfer to TAR RNA. Mol Cell Biol 32:4780-93
Nakamura, Robert L; Landt, Stephen G; Mai, Emily et al. (2012) A cell-based method for screening RNA-protein interactions: identification of constitutive transport element-interacting proteins. PLoS One 7:e48194
D'Orso, Iván; Frankel, Alan D (2010) RNA-mediated displacement of an inhibitory snRNP complex activates transcription elongation. Nat Struct Mol Biol 17:815-21
D'Orso, Iván; Frankel, Alan D (2010) HIV-1 Tat: Its Dependence on Host Factors is Crystal Clear. Viruses 2:2226-2234
D'Orso, Iván; Frankel, Alan D (2009) Tat acetylation modulates assembly of a viral-host RNA-protein transcription complex. Proc Natl Acad Sci U S A 106:3101-6
D'Orso, Ivan; Grunwell, Jocelyn R; Nakamura, Robert L et al. (2008) Targeting tat inhibitors in the assembly of human immunodeficiency virus type 1 transcription complexes. J Virol 82:9492-504
Landt, Stephen G; Tipton, Alicia R; Frankel, Alan D (2005) Localized influence of 2'-hydroxyl groups and helix geometry on protein recognition in the RNA major groove. Biochemistry 44:6547-58
Calabro, Valerie; Daugherty, Matthew D; Frankel, Alan D (2005) A single intermolecular contact mediates intramolecular stabilization of both RNA and protein. Proc Natl Acad Sci U S A 102:6849-54
Xie, Baode; Calabro, Valerie; Wainberg, Mark A et al. (2004) Selection of TAR RNA-binding chameleon peptides by using a retroviral replication system. J Virol 78:1456-63
Das, Chandreyee; Edgcomb, Stephen P; Peteranderl, Ralph et al. (2004) Evidence for conformational flexibility in the Tat-TAR recognition motif of cyclin T1. Virology 318:306-17

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