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Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype.

ABSTRACT: Osteoclasts (OCLs) in Paget's disease are markedly increased in number and size, have increased numbers of nuclei per multinucleated cell, and demonstrate increased resorption capacity and increased sensitivity to 1,25-(OH)(2)D(3), the active form of vitamin D. These cells also contain nuclear inclusions, reminiscent of those seen in paramyxovirus-infected cells, which cross-react with antibodies to measles virus nucleocapsid (MVNP) antigen. To elucidate the role of MV in the abnormal OCL phenotype of Paget's disease, we transduced normal OCL precursors with retroviral vectors expressing MVNP and the MV matrix (MVM) genes. The transduced cells were then cultured with 1,25-(OH)(2)D(3) for14 or 21 days to induce formation of OCL-like multinucleated cells. The MVNP-transduced cells formed increased numbers of multinucleated cells, which contained many more nuclei and had increased resorption capacity compared with multinucleated cells derived from empty vector-transduced (EV-transduced) and MVM-transduced or normal bone marrow cells. Furthermore, MVNP-transduced cells showed increased sensitivity to 1, 25-(OH)(2)D(3), and formed OCLs at concentrations of 1, 25-(OH)(2)D(3) that were 1 log lower than that required for normal, EV-transduced, or MVM-transduced cells. These results demonstrate that expression of the MVNP gene in normal OCL precursors stimulates OCL formation and induces OCLs that express a phenotype similar to that of pagetic OCLs. These results support a potential pathophysiologic role for MV infection in the abnormal OCL activity and morphology that are characteristic of pagetic OCLs.

SUBMITTER: Kurihara N

PROVIDER: S-EPMC289182 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature

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Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype.

Kurihara N N Reddy S V SV Menaa C C Anderson D D Roodman G D GD

The Journal of clinical investigation 20000301 5

Osteoclasts (OCLs) in Paget's disease are markedly increased in number and size, have increased numbers of nuclei per multinucleated cell, and demonstrate increased resorption capacity and increased sensitivity to 1,25-(OH)(2)D(3), the active form of vitamin D. These cells also contain nuclear inclusions, reminiscent of those seen in paramyxovirus-infected cells, which cross-react with antibodies to measles virus nucleocapsid (MVNP) antigen. To elucidate the role of MV in the abnormal OCL phenot ...[more]

PMID: 10712432

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Project description:UnlabelledThe genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P(XD) and N(TAIL) is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P(XD) F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P(XD)-to-N(TAIL) binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P(XD)-N(TAIL) interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor.ImportanceMononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼ 6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template.

Project description:Paget's disease of bone (PDB) is characterized by abnormal osteoclasts with unique characteristics that include increased sensitivity of osteoclast progenitors to 1,25(OH)2 D3 , receptor activator of NF-?B ligand (RANKL), and TNF-?; increased osteoclast numbers; and increased expression of IL-6 and several transcription factors. We recently reported that measles virus nucleocapsid protein (MVNP) plays a key role in the development of these abnormal osteoclasts. MVNP can induce the pagetic osteoclast phenotype in vitro and in vivo in TRAP-MVNP transgenic mice. However, the molecular mechanisms by which MVNP generates pagetic osteoclasts have not been determined. TANK-binding kinase 1 (TBK1) and I?B kinase-? (IKK?) are IKK family members that complex with MVNP and activate both IRF3 and NF-?B pathways. MVNP increases the amount of TBK1 protein in bone marrow monocytes (BMM). Interestingly, we found that RANKL increased TBK1 and IKK? early in osteoclast differentiation, suggesting a possible role in normal osteoclastogenesis. However, only TBK1 is further increased in osteoclasts formed by TRAP-MVNP BMM owing to increased TBK1 protein stability. TBK1 overexpression induced IL6 promoter reporter activity, and elevated endogenous IL6 mRNA and p65 NF-?B, TAF12, and ATF7 proteins in several cell lines. Overexpression of TBK1 was insufficient to induce pagetic osteoclasts from WT BMM but synergized with MVNP to increase pagetic osteoclast formation from TRAP-MVNP BMM. BX795 inhibition of TBK1 impaired MVNP-induced IL-6 expression in both NIH3T3 cells and BMM, and shRNA knockdown of Tbk1 in NIH3T3 cells impaired IL-6 secretion induced by MVNP and decreased TAF12 and ATF7, factors involved in 1,25(OH)2 D3 hypersensitivity of pagetic osteoclasts. Similarly, Tbk1 knockdown in BMM from TRAP-MVNP and WT mice specifically impaired development of the MVNP-induced osteoclast pagetic phenotype. These results demonstrate that TBK1 plays a critical role in mediating the effects of MVNP on osteoclast differentiation and on the expression of IL-6, a key contributor to the pagetic osteoclast phenotype.

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Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype.

Publications

Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype.

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