Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

Autor: Craig N. Robson, Mingzhou Chen, Jean-Claude Cortay, Ian R. Logan, Vasileia Sapountzi, Denis Gerlier
Přispěvatelé: Rollin, Bertrand
Rok vydání: 2005
Předmět:
Viral Proteins/chemistry/genetics/*metabolism
Phosphoproteins/chemistry/genetics/*metabolism
Ubiquitin-conjugating enzyme
Binding Sites/genetics
Mice
Ubiquitin
Enzyme Stability
RNA
Small Interfering
[SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology
Nucleocapsid Proteins
Measles virus/genetics/*metabolism/pathogenicity
Virus-Cell Interactions
Ubiquitin ligase
Small Interfering/genetics
Mdm2
DNA
Complementary
Immunoprecipitation
Recombinant Fusion Proteins
Ubiquitin-Protein Ligases
Immunology
Complementary/genetics
Biology
Transfection
Microbiology
Cell Line
Viral Proteins
Two-Hybrid System Techniques
Virology
Animals
Humans
Binding site
Ubiquitin-Protein Ligases/antagonists & inhibitors/chemistry/genetics/*metabolism
Binding Sites
Base Sequence
Recombinant Fusion Proteins/chemistry/genetics/metabolism
DNA
Phosphoproteins
Molecular biology
Fusion protein
Nucleoproteins
Measles virus
Hela Cells
Multiprotein Complexes
Insect Science
Phosphoprotein
biology.protein
RNA
Ubiquitin/metabolism
Nucleoproteins/chemistry/genetics/metabolism
Zdroj: Journal of Virology. 79:11824-11836
ISSN: 1098-5514
0022-538X
DOI: 10.1128/jvi.79.18.11824-11836.2005
Popis: 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 hPIRH2 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.
Databáze: OpenAIRE
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