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The Vpr accessory protein of HIV-1 induces a response similar to that of DNA damage. In cells expressing Vpr, the DNA damage sensing kinase, ATR, is activated, resulting in G 2 arrest and apoptosis. In addition, Vpr causes rapid degradation of the uracil-DNA glycosylases UNG2 and SMUG1. Although several cellular proteins have been reported to bind to Vpr, the mechanism by which Vpr mediates its biological effects is unknown. Using tandem affinity purification and mass spectrometry, we identified a predominant cellular protein that binds to Vpr as the damage-specific DNA-binding protein 1 (DDB1). In addition to its role in the repair of damaged DNA, DDB1 is a component of an E3 ubiquitin ligase that degrades numerous cellular substrates. Interestingly, DDB1 is targeted by specific regulatory proteins of other viruses, including simian virus 5 and hepatitis B. We show that the interaction with DDB1 mediates Vpr-induced apoptosis and UNG2/SMUG1 degradation and impairs the repair of UV-damaged DNA, which could account for G 2 arrest and apoptosis. The interaction with DDB1 may explain several of the diverse biological functions of Vpr and suggests potential roles for Vpr in HIV-1 replication.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr has previously been shown to bind to the cellular uracil DNA glycosylase UNG. We show here that the binding of Vpr to UNG and to the related enzyme SMUG induces their proteasomal degradation. UNG and SMUG were found to be encapsidated in Δ vpr HIV-1 virions but were significantly less abundant in vpr + virions. Δ vpr virions contained readily detectable uracil-DNA glycosylase enzymatic activity, while the activity was reduced to undetectable levels in vpr + virions. Consistent with proteasomal degradation, complexes that contained Vpr and the E3 ubiquitin ligase components Cul1 and Cul4 were detected in cell lysates. We hypothesized that the interaction of Vpr might be a means for the virus to reduce the frequency of abasic sites in viral reverse transcripts at uracil residues caused by APOBEC3-catalyzed deamination of cytosine residues. Although APOBEC3 is largely neutralized by the Vif accessory protein, residual enzyme could remain in virions that would generate uracils. In support of this, Δ vif vpr + HIV-1 produced in the presence of limited amounts of APOBEC3G was significantly more infectious than Δ vif Δ vpr virus. In Addition, vpr + HIV-1 replicated more efficiently than vpr − virus in cells that expressed limited amounts of APOBEC3G. The findings highlight the importance of cytidine deamination in the virus replication cycle and present a novel function for Vpr.

Conformational change in human IKK2 permits dimers to form higher-order oligomers that support interaction between kinase domains and promote activation through trans auto-phosphorylation., Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase., Author Summary IκB kinase (IKK) is an enzyme that quickly becomes active in response to diverse stresses on a cell. Once activated, IKK promotes an array of cellular defense processes by phosphorylating IκB, thereby promoting its degradation and liberating its partner, the pro-survival transcription factor NF-κB; NF-κB is then free to relocate to the nucleus where it can modulate gene expression. Our X-ray crystallographic studies on an active version of the human IKK2 isoform reveal that the enzyme adopts a unique open conformation that permits pairs of IKK2 enzymes to form higher order assemblies in which their catalytic domains are in close proximity. Disruption of IKK2's ability to form these assemblies, by introducing changes that interfere with the surfaces that mediate oligomerization, results in IKK2 enzymes that are greatly impaired in their ability to become activated in cells. We propose that after oligomerization the neighboring catalytic domains then phosphorylate each other as part of the activation process. Our findings also suggest that targeted small molecules might disrupt cell survival by blocking IKK2 assembly in cells.

Tumor necrosis factor receptor (TNFR) superfamily members mediate the cellular response to a wide variety of biological inputs. The responses range from cell death, survival, differentiation, proliferation, to the regulation of immunity. All these physiological responses are regulated by a limited number of highly pleiotropic kinases. The fact that the same signaling molecules are involved in transducing signals from TNFR superfamily members that regulate different and even opposing processes raises the question of how their specificity is determined. Regulatory strategies that can contribute to signaling specificity include scaffolding to control kinase specificity, combinatorial use of several signal transducers, and temporal control of signaling. In this review, we discuss these strategies in the context of TNFR superfamily member signaling.

Besides activating NFκB by phosphorylating IκBs, IKKα/IKKβ kinases are also involved in regulating metabolic insulin signaling, the mTOR pathway, Wnt signaling, and autophagy. How IKKβ enzymatic activity is targeted to stimulus-specific substrates has remained unclear. We show here that NEMO, known to be essential for IKKβ activation by inflammatory stimuli, is also a specificity factor that directs IKKβ activity toward IκBα. Physical interaction and functional competition studies with mutant NEMO and IκB proteins indicate that NEMO functions as a scaffold to recruit IκBα to IKKβ. Interestingly, expression of NEMO mutants that allow for IKKβ activation by the cytokine IL-1, but fail to recruit IκBs, results in hyperphosphorylation of alternative IKKβ substrates. Furthermore IKK's function in autophagy, which is independent of NFκB, is significantly enhanced without NEMO as IκB scaffold. Our work establishes a role for scaffolds such as NEMO in determining stimulus-specific signal transduction via the pleiotropic signaling hub IKK.

The HIV-1 accessory protein Vif (virion infectivity factor) is required for the production of infectious virions by CD4 + lymphocytes. Vif facilitates particle infectivity by blocking the inhibitory activity of APOBEC3G (CEM15), a virion-encapsidated cellular protein that deaminates minus-strand reverse transcript cytosines to uracils. We report that HIV-1 Vif forms a complex with human APOBEC3G that prevents its virion encapsidation. HIV-1 Vif did not efficiently form a complex with mouse APOBEC3G. Vif dramatically reduced the amount of human APOBEC3G encapsidated in HIV-1 virions but did not prevent encapsidation of mouse or AGM APOBEC3G. As a result, these enzymes are potent inhibitors of wild-type HIV-1 replication. The species-specificity of this interaction may play a role in restricting HIV-1 infection to humans. Together these findings suggest that therapeutic intervention that either induced APOBEC3G or blocked its interaction with Vif could be clinically beneficial.