Dataset Information

The feet of the measles virus polymerase bind the viral nucleocapsid protein at a single site.

ABSTRACT: Measles virus has a single-stranded RNA genome that is organized into a helical complex by the viral N protein. The resulting structure is termed the nucleocapsid and is traversed by the viral polymerase during RNA synthesis. The P protein, the noncatalytic subunit of the polymerase, provides the "legs and feet" that allow the polymerase to walk along its protein-RNA template. The polymerase feet are very simple three-helix bundles, only 50 amino acids in size. Previously, we have shown that these feet grasp the viral N protein during movement by attaching to a short sequence (amino acids 487-503) within the disordered and surface-exposed tail of N, causing it to fold into a helix. The result is a weak-affinity complex with a short lifetime, which would allow the polymerase to take rapid steps forward. The structure of the complex was determined using X-ray crystallography. This simple model of binding was challenged by a paper in this journal, claiming that a downstream sequence in the tail of N (amino acids 517-525) was also critical for the association. Its presence was reported to enhance the overall affinity of the polymerase feet for N by three orders of magnitude. We have, therefore, examined binding of the polymerase foot domain to amino acids 477-525 of N using quantitative biophysical techniques, and compared the results to our previous binding studies, performed using amino acids 477-505 of N. We find no evidence that the sequence downstream of amino acid 505 influences binding, validating the original single-site binding model.

SUBMITTER: Yegambaram K

PROVIDER: S-EPMC2867028 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The feet of the measles virus polymerase bind the viral nucleocapsid protein at a single site.

Yegambaram Kavestri K Kingston Richard L RL

Protein science : a publication of the Protein Society 20100401 4

Measles virus has a single-stranded RNA genome that is organized into a helical complex by the viral N protein. The resulting structure is termed the nucleocapsid and is traversed by the viral polymerase during RNA synthesis. The P protein, the noncatalytic subunit of the polymerase, provides the "legs and feet" that allow the polymerase to walk along its protein-RNA template. The polymerase feet are very simple three-helix bundles, only 50 amino acids in size. Previously, we have shown that the ...[more]

PMID: 20143306

Similar Datasets

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.

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:Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3.IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression.

Dataset Information

The feet of the measles virus polymerase bind the viral nucleocapsid protein at a single site.

Publications

The feet of the measles virus polymerase bind the viral nucleocapsid protein at a single site.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure