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Lookup NU author(s): Dr Ian Logan, Dr Vasileia Sapountzi, Professor Craig Robson
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Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged bPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Author(s): Chen, M., Cortay, J-C., Logan, I. R., Sapountzi, V., Robson, C. N., Gerlier, D.
Publication type: Article
Publication status: Published
Journal: Journal of Virology
Year: 2005
Volume: 79
Issue: 18
Pages: 11824-11836
Print publication date: 01/09/2005
ISSN (print): 0022-538X
ISSN (electronic): 1070-6321
URL: http://dx.doi.org/10.1128/JVI.79.18.11824-11836.2005
DOI: 10.1128/JVI.79.18.11824-11836.2005
PubMed id: 16140759
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