The inactivation of the prototype NF-B inhibitor, IB␣, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosincontaining protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IB␣ immune complexes. The ubiquitinated IB␣ conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-B. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IB␣ complexes sediment in the 19 S fractions, while the unmodified IB␣ sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IB␣ are critical for VCP binding, which in turn is necessary but not sufficient for IB␣ degradation; while the N-terminal domain of IB␣ is required in all three reactions, both N-and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between IB␣ and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IB␣.Transcription factor NF-B is involved in a large variety of processes, such as cell growth, transcriptional regulation, immune, inflammatory, and stress responses (reviewed in Refs. 1-4). NF-B is a homo-or heterodimer consisting of various combinations of the family members, including NFB1 (p50 and precursor p105), c-Rel, RelA, NFB2 (p52 and precursor p100), RelB, and Drosophila proteins Dorsal and Dif. Unlike many other transcription factors that are localized in the nucleus, the NF-B dimeric factor is sequestered in the cytoplasm of most cells through binding to a family of inhibitor proteins, IB. In response to extracellular stimuli, the inhibitors are partially or entirely degraded, thus liberating the DNA-binding dimer for translocation to the nucleus. The I6B family contains IB␣, IB␤, IB␥, Bcl-3, the precursor proteins p105 and p100, and the Drosophila protein Cactus. All members of the IB family contain 5-8 ankyrin motifs, thought to be involved in protein-protein interactions. It has been shown that when the precursor protein p105 is involved as the inhibitor, the processing from p105 to the active p50 occurs through the ubiquitindependent proteasome (Ub-Pr) 1 pathway, which degrades the C-terminal ankyrin-containing domain of p105 (5, 6).2 For the prototype complex that contains p50, p65, and IB␣, upon stimulation the entire NF-B complex becomes hyperphosphorylated. The induced phosphorylation of IB␣ does not lead to its immediate dissociation from the complex; rather, it signals for rapid IB␣ degradation, thus liberating the active p50⅐p65 dimer for translocation to the nucleus (7-15). We and others showed that the degradation of IB␣ is sensitive to proteasome inhibitors and is ubiquitin-dependent. Recently, it was further shown that signal-ind...

We performed radioimmunoprecipitation followed by serial immunoblots to show that, in the unstimulated Jurkat T cell line, the NF-kappa B/Rel family proteins, p80-c-Rel, p105-NF-kappa B, p65-NF-kappa B, p50-NF-kappa B and p36-I kappa B alpha, can be detected as complexes using antisera against c-Rel, p105-NF-kappa B or p65-NF-kappa B. p36-I kappa B alpha and p105, both known inhibitors of NF-kappa B function, can physically associate with NF-kappa B/Rel family members, but not with each other. In vivo and in vitro phosphorylation experiments demonstrated that NF-kappa B/Rel family members, including p105, c-Rel, p50, p65 (for the first time for p50 and p65) and p36-I kappa B alpha are also phosphoproteins. Phosphoserine and phosphothreonine residues were identified in these proteins isolated from unstimulated Jurkat cells. Both unphosphorylated and hyperphosphorylated forms of p36-I kappa B alpha were found in the complexes, suggesting that hyperphosphorylated I kappa B alpha is still capable of associating with the NF-kappa B/Rel family members. After stimulation with phorbol 12-myristate 13-acetate and phytohaemagglutinin for 10 min, p105-NF-kappa B and p50-NF-kappa B, but not p36-I kappa B, were highly phosphorylated. Phosphopeptide mapping of p105 showed that phorbol ester/phytohaemagglutinin stimulation may change p105 phosphorylation qualitatively.

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PVS-RIPO is a recombinant oncolytic poliovirus designed for clinical application to target CD155 expressing malignant gliomas and other malignant diseases. PVS-RIPO does not replicate in healthy neurons and is therefore non-pathogenic in rodent and non-human primate models of poliomyelitis. A tetrazolium salt dye-based cellular assay was developed and qualified to define the cytotoxicity of virus preparations on susceptible cells and to explore the target cell specificity of PVS-RIPO. In this assay, PVS-RIPO inhibited proliferation of U87-MG astrocytoma cells in a dose-dependent manner. However, HEK293 cells were much less susceptible to cell killing by PVS-RIPO. In contrast, the Sabin type 1 live attenuated poliovirus vaccine strain (PV(1)S) was cytotoxic to both HEK293 and U87-MG cells. The correlation between expression of CD155 and cytotoxicity was also explored using six different cell lines. There was little or no expression of CD155 and PVS-RIPO-induced cytotoxicity in Jurkat and Daudi cells. HEK293 was the only cell line tested that showed CD155 expression and resistance to PVS-RIPO cytotoxicity. The results indicate that differential cytotoxicity measured by the colorimetric assay can be used to evaluate the cytotoxicity and celltype specificity of recombinant strains of poliovirus and to demonstrate lot to lot consistency during the manufacture of viruses intended for clinical use.

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