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

Abstract

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.Measles virus (MV) is responsible for an acute childhood disease that, in 2002, infected almost 40 million infants and caused 0.8 million deaths (42). It is an enveloped virus belonging to the Mononegavirales order, the Paramyxoviridae family, and the Morbillivirus genus. The genome is a nonsegmented, negative-strand RNA. It comprises six genes, the genes for nucleoprotein (N) and phosphoprotein (P protein) and matrix (M), fusion (F), hemagglutinin (H), and large (L; polymerase) proteins, in that order, flanked by two short sequences, the leader (Le) and the trailer (Tr), which contain the genome and antigenome promoters, respectively. The MV RNA-dependent RNA polymerase comprising the L and P proteins uses a ribonucleoprotein template composed of the genomic RNA in complex with the N protein. The P protein from Paramyxoviridae acts as a bridge between L and ribonucleoprotein and has partially overlapping binding sites for both L (40) and N proteins. P has multiple functions: (i) it binds to the N-terminal domain of L (7), (ii) it acts as a molecular chaperone and stabilizes the L protein (20), (iii) it places the polymerase complex on the nucleocapsid, (iv) it allows viral transcription and replication, and (v) it prevents the illegitimate encapsidation of cellular RNA by keeping N in a monomeric N°state (22

Mapping and Functional Role of the Self-Association Domain of Vesicular Stomatitis Virus Phosphoprotein

“…These motifs are located in different positions in various P proteins, e.g., in the P protein of the family Paramyxoviridae, the coiled-coil motifs are mostly located in the C terminus (1,10,11,12,30,37,43,48,49), whereas the coiled-coil motif in respiratory syncytial virus is situated in the central part of the P protein (8) and the putative coiled-coil motif of Marburg virus and Ebola virus VP35 (family Filoviridae) is located at the N terminus which was shown to be essential for self-association (35). It is interesting that the VSV Pind also contains a predominant coiledcoil motif (12,27), which is located at the amino-terminal region as predicted by COILS prediction software (33); the region spans residues 16 to 36 (data not shown).…”

“…HeLa cells in six-well plates were infected with vTF7-3 for 1 h at a multiplicity of infection of 10, washed, and then transfected with the appropriate plasmids in the presence of Lipofectamine 2000 (Invitrogen). After 24 h of transfection, cell lysates were prepared as described elsewhere (10). In experiments to map the self-association domain of Pind protein, Myc-tagged Pind was coexpressed with HA-tagged Pind or mutants in various combinations.…”

“…Precleared supernatants of lysates were incubated with polyclonal anti-L antibody (antiserum produced from immunized rabbits by using synthesized peptides) and mixed with protein G Sepharose 4 Fast Flow medium as described above. Beads were collected and washed five times with washing buffer as described elsewhere (10). The beads were boiled in Laemmli buffer and bound proteins were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and detected by Western blotting using antiMyc monoclonal antibody sc-40 (Santa Cruz), anti-HA antibody (Sigma), or anti-Flag antibody (Santa Cruz).…”

Sequence of Events in Measles Virus Replication: Role of Phosphoprotein-Nucleocapsid Interactions

“…Parental Vero, si1 and si2 cells were infected at indicated MOI with recombinant viruses with or without addition of 10 g/ml of fusion inhibitor peptide z-fFG to prevent syncytium formation. Viral protein expression was determined by flow cytometry analysis of cells labeled with the Y503 anti-F monoclonal antibody and/or GFP detection, detection of the expression of viral N (cl25 antibody), P (49.21 antibody), HA-P 2 (anti-HA antibody; Sigma), Flag-P 1 (anti-Flag antibody; Sigma), and cellular GAPDH (MAb374 antibody; Chemicon) protein by Western blotting revealed by chemiluminescence as detailed previously (22,23). Unexpectedly, when the P protein was tagged with the HA peptide, its recognition by 49.21 monoclonal antibody anti-P was reduced.…”

Pirh2 interacts with and ubiquitylates signal recognition particle receptor β subunit

“…phosphoprotein (3), N-terminal kinase-like proteinbinding protein 1 (NTKL-BP1) (22), and calmodulin (5). Recently, it has been reported that histone deacetylase 1 (HDAC1) and the ε-subunit of coatomer complex (ε-COP) were ubiquitylated and subsequently degraded by Pirh2 (13,14).…”