Modular Organization of Rabies Virus Phosphoprotein

Gérard, Francine C A; Ribeiro, Euripedes de Almeida; Leyrat, C.; Ivanov, Ivan; Blondel, Danielle; Longhi, Sonia; Ruigrok, Rob W.H.; Jamin, Marc

doi:10.1016/j.jmb.2009.03.061

Cited by 101 publications

(142 citation statements)

References 119 publications

(217 reference statements)

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“…This has already been well illustrated in the case of the P proteins from measles (44) and Sendai viruses (83, 99 -102) as well as for the phosphoproteins from Rhabdoviridae members (103)(104)(105)(106)(107), and, more generally, for viral proteins targeted by large scale structural genomics projects (for examples see Refs. 108 -116).…”

Section: Discussionmentioning

confidence: 71%

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

Habchi

Blangy

Mamelli

et al. 2011

Journal of Biological Chemistry

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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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Section: Discussionmentioning

confidence: 71%

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

Habchi

Blangy

Mamelli

et al. 2011

Journal of Biological Chemistry

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165

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“…These phosphoproteins are modular proteins that have an N-terminal domain that keeps newly produced N (called N 0 ) in a soluble, RNAfree form, a central oligomerization domain and a C-terminal domain that binds to N-RNA. The three domains are connected by two intrinsically disordered regions (10,13). The atomic structures of the oligomerization domains of the phosphoproteins of VSV and Sendai virus have been determined, and it was found that these structures were quite different (6,21).…”

mentioning

confidence: 99%

“…The sequence analysis of rabies virus P suggests that the oligomerization domain stretches from amino acid residues 92 to 131 (10). The DNA corresponding to residues 91 to 133 was cloned into a pET22b vector and expressed in the Escherichia coli BL21 (DE3-RIL) strain as a His tag fusion protein.…”

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confidence: 99%

“…The protein was purified by nickel resin and size-exclusion chromatography (S75). In solution the protein behaves as a dimer with a molecular mass of 13.9 kDa (monomer, 6,501 Da) (10). Monomers or higher-order oligomers have never been observed.…”

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confidence: 99%

“…The largest functional difference between the three oligomerization domains is that in the rabies virus P dimer the N-terminal end that is linked to the N 0 binding domain is positioned next to the C-terminal end that is linked to the N-RNA binding domain, whereas in both the VSV and the Sendai virus structures the N-and C-terminal ends are at opposite ends of the oligomerization domain. However, this difference may not have biological consequences because for all three phosphoproteins the N 0 and the N-RNA binding domains are connected to the oligomerization domain by extensive disordered regions (10).…”

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confidence: 99%

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Structure of the Dimerization Domain of the Rabies Virus Phosphoprotein

Ivanov

Crépin

Jamin

et al. 2010

J Virol

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The crystal structure of the dimerization domain of rabies virus phosphoprotein was determined. The monomer consists of two ␣-helices that make a helical hairpin held together mainly by hydrophobic interactions. The monomer has a hydrophilic and a hydrophobic face, and in the dimer two monomers pack together through their hydrophobic surfaces. This structure is very different from the dimerization domain of the vesicular stomatitis virus phosphoprotein and also from the tetramerization domain of the Sendai virus phosphoprotein, suggesting that oligomerization is conserved but not structure.Rabies virus is a negative-strand RNA virus of the Rhabdovirus family. Its genomic RNA is encapsidated by numerous copies of the viral nucleoprotein (N) that binds to the sugar phosphate backbone of the RNA with a stoichiometry of nine nucleotides per N protomer (1). RNA replication and transcription take place on this N-RNA template (2) and are catalyzed by the viral RNA-dependent RNA polymerase (L). L binds to the N-RNA template with the help of the polymerase cofactor, the phosphoprotein (P) (7). The phosphoproteins of the Rhabdoviridae and of the Paramyxoviridae are oligomers (5, 8); P of the Rhabdoviridae (rabies virus and vesicular stomatitis virus [VSV]) form dimers (6, 9), whereas P of the Paramyxoviridae (Sendai virus) form tetramers (21). These phosphoproteins are modular proteins that have an N-terminal domain that keeps newly produced N (called N 0 ) in a soluble, RNAfree form, a central oligomerization domain and a C-terminal domain that binds to N-RNA. The three domains are connected by two intrinsically disordered regions (10, 13). The atomic structures of the oligomerization domains of the phosphoproteins of VSV and Sendai virus have been determined, and it was found that these structures were quite different (6, 21). Because the secondary structure prediction of the oligomerization domain of rabies virus P was different again from that of Sendai virus and VSV (10), we decided to determine its structure.The sequence analysis of rabies virus P suggests that the oligomerization domain stretches from amino acid residues 92 to 131 (10). The DNA corresponding to residues 91 to 133 was cloned into a pET22b vector and expressed in the Escherichia coli BL21 (DE3-RIL) strain as a His tag fusion protein. The protein was purified by nickel resin and size-exclusion chromatography (S75). In solution the protein behaves as a dimer with a molecular mass of 13.9 kDa (monomer, 6,501 Da) (10). Monomers or higher-order oligomers have never been observed.The protein was crystallized at 20°C in 50 mM sodium cacodylate (pH 6.5)-10 mM MgSO 4 plus 2 M (NH 4 ) 2 SO 4 . Diffraction data on frozen crystals were obtained on beam lines ID14-4 and BM14 (ESRF, Grenoble, France). The crystals diffracted to a 1.5-Å resolution and belong to space group I4 1 22 with one dimer in the asymmetric unit. The phases were solved by the Sulfur-MAD method (Table 1). Integration and scaling of the data were performed with XDS (12). Three methionine sites ...

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