Interaction of the C-Terminal Domains of Sendai Virus N and P Proteins: Comparison of Polymerase-Nucleocapsid Interactions within the Paramyxovirus Family

Houben, Klaartje; Marion, Dominique; Tarbouriech, Nicolas; Ruigrok, Rob W.H.; Blanchard, Laurence

doi:10.1128/jvi.00338-07

Cited by 112 publications

(118 citation statements)

References 45 publications

(46 reference statements)

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“…For measles and Sendai viruses, a weak-affinity protein complex with a short lifetime is known to result. 8,31 This is consistent with rapid movement of the polymerase along its template during RNA synthesis.…”

Section: Discussionsupporting

confidence: 54%

“…This study re-emphasizes some of the common principles which govern operation of the replication machinery in the Paramyxovirinae; a family that includes measles, mumps, and Sendai viruses. Although the intermolecular forces which drive the polymerase feet to interact with the nucleocapsid differ in these viruses, 7,8,[30][31][32][33] in all three cases, the interaction involves the coupled binding and folding of protein domains. For measles and Sendai viruses, a weak-affinity protein complex with a short lifetime is known to result.…”

Section: Discussionmentioning

confidence: 99%

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

Yegambaram

Kingston

2010

Protein Science

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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.

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Section: Discussionsupporting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

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

Yegambaram

Kingston

2010

Protein Science

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“…For measles virus (MV) and Sendai virus (SeV), which belong to the genera Morbillivirus and the Respirovirus, respectively, of the family Paramyxoviridae, the C-terminal end of P forms a small and dynamic three-helical bundle that stabilizes upon binding to the disordered C-terminal domain of N, termed N TAIL (19,20,23). For the Rhabdoviridae (rabies virus and VSV), the C-terminal domain of P that binds to N is much larger and more structured than those of MV and SeV (30,36).…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

Galloux

Tarus

Blazevic

et al. 2012

J Virol

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The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (P CTD ) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (N NTD ) as a P binding domain. A biochemical characterization of the P CTD and molecular modeling of the N NTD allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the P CTD interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.H uman respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract infections in newborn children worldwide (7). It infects close to 100% of infants within the first 2 years of life and is the main cause of bronchiolitis. Also, bovine respiratory syncytial virus (BRSV), which is very similar to its human counterpart, is a major cause of respiratory disease in calves, resulting in substantial economic losses to the cattle industry worldwide (49). RSV belongs to the genus Pneumovirus of the family Paramyxoviridae and the order Mononegavirales (7). The viral genome consists of a nonsegmented ϳ15-kb RNA of negative polarity which encodes 11 proteins. As for all the members of the Mononegavirales, the genomic RNA of RSV is always tightly bound by the viral nucleoprotein (N) and maintained as a helical N-RNA ribonucleoprotein (RNP) complex (9). The RNP is used as the template for transcription and replication by the RNAdependent RNA polymerase (RdRp) complex, consisting of L (large protein) and its cofactor P (phosphoprotein). Whereas N, P, and L are sufficient to mediate viral replication (15, 50), transcription activity requires L, P, and the M2-1 protein, which functions as a processivity polymerase cofactor (8).The specific recognition of the viral N-RNA matrix by the RdRp constitutes a prerequisite for vir...

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“…As a result, IDP interfaces make more hydrophobic contacts (33% for IDPs and 22% for ordered proteins), whereas ordered interfaces make more polar interactions (122). A notable exception is provided by the SeV N TAIL -P XD complex that mainly relies on polar contacts and is therefore impaired by high salt concentrations (83).…”

Section: Structural Models Of Henipavirus N Tail -P Xd Complexes and mentioning

confidence: 99%

“…131 and references cited therein). On the other hand, SeV (83) and Henipavirus would provide examples of the second scenario, with K D values in the M range. These findings would support a cartwheeling mechanism for the polymerase complex of Henipavirus, as already proposed for other Paramyxoviridae members (132,133).…”

Section: Structural Models Of Henipavirus N Tail -P Xd Complexes and mentioning

confidence: 99%

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

Habchi

Blangy

Mamelli

et al. 2011

Journal of Biological Chemistry

165

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The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N TAIL ) are both intrinsically disordered. Here we show that Henipavirus N TAIL domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P XD ) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N TAIL and P XD form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K D in the M range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P XD triggers an increase in the ␣-helical content of N TAIL . Using fluorescence spectroscopy, we show that P XD has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N TAIL domain, thus arguing for the lack of stable contacts between the C termini of N TAIL and P XD . Finally, we present a tentative structural model of the N TAIL -P XD interaction in which a short, order-prone region of N TAIL (␣-MoRE; amino acids 473-493) adopts an ␣-helical conformation and is embedded between helices ␣2 and ␣3 of P XD , leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N TAIL -P XD interaction as a valuable target for rational antiviral approaches.

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Interaction of the C-Terminal Domains of Sendai Virus N and P Proteins: Comparison of Polymerase-Nucleocapsid Interactions within the Paramyxovirus Family

Cited by 112 publications

References 45 publications

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

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

Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

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