Viruses must compete with their host cell for macromolecular machineries at many levels after infection and then use the cell’s resources for virus replication, transcription, processing and transport of mRNAs, and protein synthesis, among others. Transcription is one example of the cell being co-opted by HIV-1, the causative agent of AIDS, where the viral encoded transcriptional regulator Tat is essential for viral replication and persistence infection. Tat recruits the transcription cellular machinery to the viral promoter, including the transcription elongation factor P-TEFb, to relieve a block in the elongation step of transcription. Besides regulating its own transcriptional program, Tat and other accessory proteins modulate the host-cell gene expression profile, using unknown mechanisms, thus impacting on the progression and pathogenesis of the disease.
Our long-term goals are to understand:
- The molecular basis of HIV and lentiviral transcription
- Why HIV evolved an RNA structure to direct transcription of its genome
- How HIV accessory and regulatory factors de-regulate the host-cell expression profile
- How HIV affects the host-cell chromatin and how it impacts on the pathogenesis of AIDS
- What selective pressures shaped the evolution of co-evolving genes in host-viral transcription complexes
- How these events dictate entry into viral latency or persistent infection
Switching Transcription Initiation into Elongation at the HIV Promoter
We have recently shown that Tat and P-TEFb are early loaded into the viral promoter and then transferred to the nascent RNA (TAR), and we have defined a critical role for TAR in the process of switching transcription initiation into elongation by ejecting an inhibitory snRNP complex to activate the P-TEFb kinase (see below). These discoveries open new paradigms in the process of viral transcription regulation and understanding the mechanistic details of this switch is critical for the rationale design of alternative therapies to combat viral replication and latency. Publications: D’Orso and Frankel, 2010a.
A Non-Coding RNA Governs the Escape into Productive Elongation at the HIV Promoter
P-TEFb, the positive transcription elongation factor, exists also as a catalytically inactive form when it is assembled into an RNA-protein complex (7SK snRNP) with the 7SK non-coding snRNA, and the Hexim1, Larp7 and Mepce proteins. We have recently shown that Tat highjacks the 7SK snRNP complex by competing off Hexim1 and assembling into the snRNP to form the Tat:7SK snRNP complex at the HIV promoter. Currently, we are working to dissect out the mechanistic basis of Tat:7SK snRNP complex assembly and what is the role of TAR and/or cellular protein complexes in the disassembly of 7SK snRNP to activate the viral elongation program.Publications: D’Orso and Frankel, 2010a.
Uncoupling Steps in the Cycle of HIV Transcription
By exploiting a combination of cell-based assays with a systematic single-site mutagenesis approach of Tat we have uncoupled steps in the cycle of HIV transcription activation. For the first time we have assigned the requirement of Tat residues in the steps of promoter binding and recruitment to the nascent transcript as the TAR stem-loop emerges from the RNA polymerase II complex. We are working towards obtaining a high-resolution map for the conversion of the Tat:7SK snRNP complex into the active form of the elongation complex at the HIV promoter. By uncovering the structural and molecular basis of the transition from an inactive to an active form of transcription we will block viral replication and pursue new approaches to purge the cell from the viral latent reservoirs. Publications: D’Orso and Frankel, 2009; D’Orso et al, 2010b.
Genome-Wide Location Scans of Tat Underlying Gene Expression Profile Alterations
Besides its role in HIV-1 transcription, Tat alters the homeostasis of the cell by repressing or activating the expression of a subset of cellular genes using unknown mechanism(s). A major limitation of previous studies in this area was the lack of an approach to examine the assembly of Tat into HIV-1 and cellular transcription complexes to modulate viral and cellular gene expression. We are working towards implementing a systems level approach to globally tackle this problem in an unbiased and more comprehensive manner. We are aiming at understanding basic mechanisms whereby viral factors “shape” the host-cell chromatin to ultimately uncover connections between viral infection with AIDS pathogenesis and disease progression. Publications: in progress.