1887

Journal of General Virology header logo

Volume 88, Issue 1

The nine C-terminal amino acids of the respiratory syncytial virus protein P are necessary and sufficient for binding to ribonucleoprotein complexes in which six ribonucleotides are contacted per N protein protomer Free

Abstract

The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in . Pull-down assays performed with a series of truncated forms of P fused to glutathione -transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100–160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161–241) fused to GST pulled down exclusively RNA–N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22 900 and 24 820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5′ and 3′ ends.

  • Received:
  • Accepted:
  • Published Online:
SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.82282-0
2007-01-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/jgv/88/1/196.html?itemId=/content/journal/jgv/10.1099/vir.0.82282-0&mimeType=html&fmt=ahah

References

  1. Asenjo A., Villanueva N. 2000; Regulated but not constitutive human respiratory syncytial virus (HRSV) P protein phosphorylation is essential for oligomerization. FEBS Lett 467:279–284 [CrossRef]
     [Google Scholar]
  2. Barik S., McLean T., Dupuy L. C. 1995; Phosphorylation of Ser232 directly regulates the transcriptional activity of the P protein of human respiratory syncytial virus: phosphorylation of Ser237 may play an accessory role. Virology 213:405–412 [CrossRef]
     [Google Scholar]
  3. Bhella D., Ralph A., Murphy L. B., Yeo R. P. 2002; Significant differences in nucleocapsid morphology within the Paramyxoviridae . J Gen Virol 83:1831–1839
     [Google Scholar]
  4. Blanchard L., Tarbouriech N., Blackledge M., Timmins P., Burmeister W. P., Ruigrok R. W. H., Marion D. 2004; Structure and dynamics of the nucleocapsid-binding domain of the Sendai virus phosphoprotein in solution. Virology 319:201–211 [CrossRef]
     [Google Scholar]
  5. Castagné N., Barbier A., Bernard J., Rezaei H., Huet J.-C., Henry C., Da Costa B., Eléouët J.-F. 2004; Biochemical characterization of the respiratory syncytial virus P–P and P–N protein complexes and localization of the P protein oligomerization domain. J Gen Virol 85:1643–1653 [CrossRef]
     [Google Scholar]
  6. Collins P. L., Hill M. G., Camargo E., Grosfeld H., Chanock R. M., Murphy B. R. 1995; Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5′ proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. Proc Natl Acad Sci U S A 92:11563–11567 [CrossRef]
     [Google Scholar]
  7. Collins P. L., Chanock R. M., Murphy B. R. 2001; Respiratory syncytial virus. In Fields Virology , 4th edn. pp  1443–1485 Edited by Knipe D. M., Howley P. M. Philadelphia, PA: Lippincott Williams & Wilkins;
     [Google Scholar]
  8. Conway J. F., Steven A. C. 1999; Methods for reconstructing density maps of “single” particles from cryoelectron micrographs to subnanometer resolution. J Struct Biol 128:106–118 [CrossRef]
     [Google Scholar]
  9. Cowton V. M., Fearns R. 2005; Evidence that the respiratory syncytial virus polymerase is recruited to nucleotides 1 to 11 at the 3′ end of the nucleocapsid and can scan to access internal signals. J Virol 79:11311–11322 [CrossRef]
     [Google Scholar]
  10. Curran J., Marq J.-B., Kolakofsky D. 1995; An N-terminal domain of the Sendai paramyxovirus P protein acts as a chaperone for the NP protein during the nascent chain assembly step of genome replication. J Virol 69:849–855
     [Google Scholar]
  11. García J., García-Barreno B., Vivo A., Melero J. A. 1993; Cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22K protein. Virology 195:243–247 [CrossRef]
     [Google Scholar]
  12. García-Barreno B., Delgado T., Melero J. A. 1996; Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusions. J Virol 70:801–808
     [Google Scholar]
  13. Grosfeld H., Hill M. G., Collins P. L. 1995; RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA. J Virol 69:5677–5686
     [Google Scholar]
  14. Johansson K., Bourhis J.-M., Campanacci V., Cambillau C., Canard B., Longhi S. 2003; Crystal structure of the measles virus phosphoprotein domain responsible for the induced folding of the C-terminal domain of the nucleoprotein. J Biol Chem 278:44567–44573 [CrossRef]
     [Google Scholar]
  15. Khattar S. K., Yunus A. S., Samal S. K. 2001a; Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N–P and P–L interactions using a minigenome system. J Gen Virol 82:775–779
     [Google Scholar]
  16. Khattar S. K., Yunus A. S., Collins P. L., Samal S. K. 2001b; Deletion and substitution analysis defines regions and residues within the phosphoprotein of bovine respiratory syncytial virus that affect transcription, RNA replication, and interaction with the nucleoprotein. Virology 285:253–269 [CrossRef]
     [Google Scholar]
  17. Kingston R. L., Hamel D. J., Gay L. S., Dahlquist F. W., Matthews B. W. 2004; Structural basis for the attachment of a paramyxoviral polymerase to its template. Proc Natl Acad Sci U S A 101:8301–8306 [CrossRef]
     [Google Scholar]
  18. Kolakofsky D., Le Mercier P., Iseni F., Garcin D. 2004; Viral DNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis. Virology 318:463–473 [CrossRef]
     [Google Scholar]
  19. Kolakofsky D., Roux L., Garcin D., Ruigrok R. W. H. 2005; Paramyxovirus mRNA editing, the ‘rule of six’ and error catastrophe: a hypothesis. J Gen Virol 86:1869–1877 [CrossRef]
     [Google Scholar]
  20. Llorente M. T., García-Barreno B., Calero M., Camafeita E., López J. A., Longhi S., Ferrón F., Varela P. F., Melero J. A. 2006; Structural analysis of the human respiratory syncytial virus phosphoprotein: characterization of an α -helical domain involved in oligomerization. J Gen Virol 87:159–169 [CrossRef]
     [Google Scholar]
  21. Loboda A. V., Ackloo S., Chernushevich I. V. 2003; A high-performance matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometer with collisional cooling. Rapid Commun Mass Spectrom 17:2508–2516 [CrossRef]
     [Google Scholar]
  22. Longhi S., Receveur-Bréchot V., Karlin D., Johansson K., Darbon H., Bhella D., Yeo R., Finet S., Canard B. 2003; The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein. J Biol Chem 278:18638–18648 [CrossRef]
     [Google Scholar]
  23. Lu B., Brazas R., Ma C.-H., Kristoff T., Cheng X., Jin H. 2002; Identification of temperature-sensitive mutations in the phosphoprotein of respiratory syncytial virus that are likely involved in its interaction with the nucleoprotein. J Virol 76:2871–2880 [CrossRef]
     [Google Scholar]
  24. Ludtke S. J., Baldwin P. R., Chiu W. 1999; eman: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128:82–97 [CrossRef]
     [Google Scholar]
  25. Mallipeddi S. K., Lupiani B., Samal S. K. 1996; Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N–P interaction using a two-hybrid system. J Gen Virol 77:1019–1023 [CrossRef]
     [Google Scholar]
  26. Mason S. W., Aberg E., Lawetz C., DeLong R., Whitehead P., Liuzzi M. 2003; Interaction between human respiratory syncytial virus (RSV) M2-1 and P proteins is required for reconstitution of M2-1-dependent RSV minigenome activity. J Virol 77:10670–10676 [CrossRef]
     [Google Scholar]
  27. Mavrakis M., McCarthy A. A., Roche S., Blondel D., Ruigrok R. W. H. 2004; Structure and function of the C-terminal domain of the polymerase cofactor of rabies virus. J Mol Biol 343:819–831 [CrossRef]
     [Google Scholar]
  28. Mavrakis M., Méhouas S., Réal E., Iseni F., Blondel D., Tordo N., Ruigrok R. W. H. 2006; Rabies virus chaperone: identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific RNA. Virology 349:422–429 [CrossRef]
     [Google Scholar]
  29. Mazumder B., Adhikary G., Barik S. 1994; Bacterial expression of human respiratory syncytial viral phosphoprotein P and identification of Ser237 as the site of phosphorylation by cellular casein kinase II. Virology 205:93–103 [CrossRef]
     [Google Scholar]
  30. Navarro J., López-Otín C., Villanueva N. 1991; Location of phosphorylated residues in human respiratory syncytial virus phosphoprotein. J Gen Virol 72:1455–1459 [CrossRef]
     [Google Scholar]
  31. Samal S. K., Collins P. L. 1996; RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end. J Virol 70:5075–5082
     [Google Scholar]
  32. Sánchez-Seco M. P., Navarro J., Martínez R., Villanueva N. 1995; C-Terminal phosphorylation of human respiratory syncytial virus P protein occurs mainly at serine residue 232. J Gen Virol 76:425–430 [CrossRef]
     [Google Scholar]
  33. Slack M. S., Easton A. J. 1998; Characterization of the interaction of the human respiratory syncytial virus phosphoprotein and nucleocapsid protein using the two-hybrid system. Virus Res 55:167–176 [CrossRef]
     [Google Scholar]
  34. Tarbouriech N., Curran J., Ruigrok R. W. H., Burmeister W. P. 2000; Tetrameric coiled coil domain of Sendai virus phosphoprotein. Nat Struct Biol 7:777–781 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.82282-0
Loading
The nine C-terminal amino acids of the respiratory syncytial virus protein P are necessary and sufficient for binding to ribonucleoprotein complexes in which six ribonucleotides are contacted per N protein protomer
J. Gen. Virol. 88, 196 (2007); https://doi.org/10.1099/vir.0.82282-0
/content/journal/jgv/10.1099/vir.0.82282-0
/content/journal/jgv/10.1099/vir.0.82282-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error