The fundamental interest of the lab is to understand how protein complexes are assembled in the context of RNA regulatory machines. This requires a detailed knowledge of the interactions that contribute to RNA-binding specificity, as well as defining the protein-protein interactions that modulate binding specificity and complex formation. Our studies focus largely on the biochemistry, structure, and mechanism of two model systems from HIV, Tat and Rev, that promote viral transcription and mRNA export, respectively. Some of our efforts are aimed at identifying new Tat- and Rev-host protein complexes, including factors that regulate the assembly of transcription complexes and others that may assist in trafficking viral RNAs to the ribosome or sites of virus particle assembly. These complexes are defining new molecular surfaces that may represent novel targets for HIV inhibitors. Much of this work is being conducted in the context of the HARC (HIV Accessory and Regulatory Complexes) Center, aimed at understanding the structural basis of important regulatory steps in the virus life cycle.
Rev binds to a large RNA, the RRE (Rev response element) located in the Env region of the genome, and mediates export of unspliced viral mRNAs from the nucleus to the cytoplasm. The Rev-RRE interaction forms a cooperative, high-affinity hexameric complex in which distinct surfaces of an alpha helical arginine-rich motif (ARM) from each of the individual Rev subunits are used to recognize several different sites on the RRE. This structural adaptability is one mechanism by which the virus apparently has evolved to optimize use of its small genome. We solved the crystal structure of a Rev dimer and its oligomeric arrangement, which shows how Rev acts as an adapter between the viral RNA and host export machinery. The importance of each of these protein-protein interfaces is being examined in the context of virus replication. We have obtained protein-RNA co-crystals and also have assembled the entire export complex with the Rev-RRE hexamer and the Crm1-RanGTP nuclear export receptor and are examining its structure using cryo-electron microscopy and crystallography. In addition, we have identified several novel host factors that interact with Rev as part of a large proteomics effort, and are probing the importance of these proteins in the viral life cycle. The oligomeric interfaces, protein-RNA interaction, and Rev-host protein interaction surfaces are being evaluated as possible targets for new HIV inhibitors.
Rev hexamer generated using the arrangement of three Rev dimers.
Adapted from Daugherty et al, 2010
Rev-host protein interactions identified from HIV proteomics.
Adapted from Jäger et al, 2011
Tat is a viral transcription factor that binds to a small RNA hairpin, TAR, located at the 5’ end of viral mRNAs and enhances transcription elongation from the viral promoter. Tat also possesses an ARM domain that recognizes the RNA, and does so in conjunction with P-TEFb, a key host elongation factor composed of Cyclin T1 and Cdk9 subunits. We have discovered that Tat, along with an inactive form of P-TEFb bound to an inhibitory 7SK snRNP complex, are recruited to the promoter prior to transcription and are later transferred to the RNA when TAR is transcribed, ejecting the inhibitory snRNP and activating the P-TEFb kinase to phosphorylate the CTD of RNA Pol II. As with Rev, we have identified several novel host factors that interact with Tat through proteomics, and are probing the roles of these proteins in viral transcription. We also have uncovered connections between the Tat and Rev regulatory circuits and are examining how the two functions have co-evolved in the virus context.
New mechanism for Tat activation. Synthesis of TAR leads to ejection of the 7SK RNP. Adapted from D'Orso and Frankel, 2011
Tat-host protein interactions identified from HIV proteomics.
Adapted from Jäger et al, 2011