Murr1 Inhibition of NF-kappaB-dependent HIV Transcription

Tom Huxford, San Diego State University Research Foundation
Biomedical and Clinical Sciences

Background: In addition to the genes encoded by HIV on the viral genome, HIV depends upon host cellular factors and processes throughout its life cycle. The development of drug therapies that interfere with these cellular processes is vital as they represent immutable targets. HIV relies heavily upon host cell machinery in activating expression of latent viral DNA that is integrated within transcriptionally silent regions of heterochromatin. The virus accomplishes this by using the strong transcriptional potential of NF-kappaB, a mastertranscriptionfactor thatbecomesactivated in response to signals produced by normal viral infection of immune cells. The transition of NF-kappaB from its inactive to active state requires the phosphorylation and ubiquitin-dependent proteolysis of its inhibitor, IkappaB. The small protein Murr1 has been shown to interfere with this process by interacting with inactive NF-kappaB/IkappaBalpha complexes and impeding ubiquitinylation of the inhibitor. As a result of this activity, Murr1 disrupts the ability of NF-kappaB to activate transcription from the long terminal repeat (LTR) HIV promoter. Structural and biochemical characterization of these factors could lead to the discovery of a new strategy, complementary to the existing therapies aimed at inhibition of viral enzymes, to maintain HIV in its latent state by targeting host cell factors.

Methods: We have successfully expressed and purified to homgeneity milligram amounts of recombinant Murr1 protein as well as the NF-kappaB p50 and p65 subunits and IkappaBalpha. The folded structure of Murr1 will be probed by controlled proteolysis and mass spectrometry. As Murr1 was original cloned and characterized as a gene involved in copper metabolism, the metal-binding properties of Murr1 will be assessed. Finally, the ability of Murr1 to undergo phosphorylation by protein kinase CK2 (casein kinase II) will be quantitated and its effects on Murr1 structure and function will be assessed. Next, three independent methods for determining Murr1 binding affinity to inactive NF-kappaB/ IkappaBalpha complexes will be employed. A new in vitro functional assay will be developed which directly measures the ability of Murr1 to inhibit ubiquitinylation of NF-kappaB/IkappaBalpha. Finally, the three-dimensionalfoldedstructure ofMurr1 bothinits free state and in complex with NF-kappaB/IkappaBalpha will be determined by x-ray crystallography. Structure determination and analysis together with binding and ubiquitinylation assay data will reveal one mechanism employed by nature to disrupt the ability of NF-kappaB to activate expression of new HIV. It will serve as a template for future studies aimed at small molecule intervention of this process with the aim of rendering latent reservoirs of HIV incapable of reproducing.

Results: Under our reaction conditions, we do not detect an interaction between recombinant Murr1 and NF-kappaB, either alone or in complex with IkappaBalpha. We are investigating the structure of the native and recombinant Murr1 proteins to account for the difference between the observations made in cell-based assays and our reductionist in vitro biochemical system. Recent reports indicate that Murr1 is a member of a family of conserved genes that share a structural motif that enables them to interact with NF-kappaB. We are currently focused on identifying the basis of this structural motif so that we can further pursue understanding the effect of Murr1 on NF-kappaB-dependent HIV viral transcription.