Inhibition of ubiquitination and stabilization of human ubiquitin E3 ligase PIRH2 by measles virus phosphoprotein.
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 co-immunoprecipitated 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 co-immunoprecipitation 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.
Chen
Mingzhou
M
Immunité & Infections Virales, CNRS--Univ-Lyon 1 UMR 5537, IFR Laennec, 69372 Lyon Cedex 08, France.
Cortay
Jean-Claude
JC
Logan
Ian R
IR
Sapountzi
Vasileia
V
Robson
Craig N
CN
Gerlier
Denis
D
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D006367:HeLa Cells; D006801:Humans; D008459:Measles virus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D051379:Mice; D046912:Multiprotein Complexes; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D046912:Multiprotein Complexes; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; C055859:P protein, Sendai virus; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D014764:Viral Proteins; C058314:nucleoprotein, Measles virus; C485369:RCHY1 protein, human; D044767:Ubiquitin-Protein Ligases