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The Nucleoprotein and Phosphoprotein of Measles Virus - PubMed

  • ️Tue Jan 01 2019

Review

The Nucleoprotein and Phosphoprotein of Measles Virus

Serafima Guseva et al. Front Microbiol. 2019.

Abstract

Measles virus is a negative strand virus and the genomic and antigenomic RNA binds to the nucleoprotein (N), assembling into a helical nucleocapsid. The polymerase complex comprises two proteins, the Large protein (L), that both polymerizes RNA and caps the mRNA, and the phosphoprotein (P) that co-localizes with L on the nucleocapsid. This review presents recent results about N and P, in particular concerning their intrinsically disordered domains. N is a protein of 525 residues with a 120 amino acid disordered C-terminal domain, Ntail. The first 50 residues of Ntail extricate the disordered chain from the nucleocapsid, thereby loosening the otherwise rigid structure, and the C-terminus contains a linear motif that binds P. Recent results show how the 5' end of the viral RNA binds to N within the nucleocapsid and also show that the bases at the 3' end of the RNA are rather accessible to the viral polymerase. P is a tetramer and most of the protein is disordered; comprising 507 residues of which around 380 are disordered. The first 37 residues of P bind N, chaperoning against non-specific interaction with cellular RNA, while a second interaction site, around residue 200 also binds N. In addition, there is another interaction between C-terminal domain of P (XD) and Ntail. These results allow us to propose a new model of how the polymerase binds to the nucleocapsid and suggests a mechanism for initiation of transcription.

Keywords: Cryo-EM; NMR; RNA binding; X-ray crystallography; measles virus; nucleoprotein; phosphoprotein.

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Figures

Figure 1
Figure 1

Schemes for the genes and for the nucleoprotein (N) and phosphoprotein (P) of measles virus. (A) The viral RNA of measles virus with its genes; The second gene codes for 3 proteins, P, C, and V and the rest of the genes code for one protein. Throughout this review the structures of N are shown in blue, for P in red and in Figure 8 the polymerase of VSV is shown in yellow. (B) The schemes of N and P proteins show the structured parts with colored bars and the disordered parts with a line. (C) The structured parts of N and P. Left, for the structure of N0P we used the PDB (Protein Data Bank) number 4CO6 and we show the Narm (left) and the Carm (right) with dash; middle, for the tetramerization domain of P we used PDB 3ZDO and right, for the complex between the linear helical motif of Ntail and the X domain of P we used PDB 1T6O.

Figure 2
Figure 2

Scheme for N0P and the assembly of nucleocapsid-like particles (NCLP). (A) The N0P complex is made from one full N protomer with the Narm and the Carm that continues in the Ntail plus a tetramer of P. Close to the end of the Ntail there is the helical motif that could bind to the X domain of P. For this picture we show only one full Ptail. The two N-terminal helices of Ptail bind N0 and for the crystal structures of N0P all the Narm, Carm plus Ntail and almost all of P (only the first 2 helices of P are binding to N0) were cut away. (B) Scheme of the assembly of the NCLP with N0P with α1/2 of P and synthesized RNA (Milles et al., 2016). When the RNA binds N0, the two helices of P dissociates from the N0 and the monomer will assemble to the NCLP. In the NCLP, the N-terminal end of the Narm forms a helix that binds to the protomer i-1 and the Carm binds to protomer i+1. In the NCLP the 5′ and 3′ protomers both have one empty site.

Figure 3
Figure 3

Role of Ntail in the nucleocapsid helix. (A) Tight helical NCLP with cleaved N with negative staining EM on the left and with the near-atomic structure on the right. The figure was made using PDB 4UFT. (B) Loose helical NCLP with full N with negative staining EM at left and with near-atomic structure with several tails. One Ntail with its helical motif is highlighted in yellow.

Figure 4
Figure 4

Kinetics of the assembly of NCLP by NMR, fluorescence and EM. Just like in Figure 2B, when RNA was given to the N0P complex, NCLP assembly could followed by NMR in real time because the NMR signals for the P peptide appear over time, by fluorescence anisotropy using fluorescein amidite-labeled RNAs, and by negative staining EM showing the nucleocapsids. For (A) we used PDB number 4CO6 for the N0P complex and PDB 4UFT for the NCLP, (B) is reprinted from Milles et al. (2016); Copyright 2016 Wiley-VCH Verlag GmbH Co. KGaA, used with permission.

Figure 5
Figure 5

Structure of the 5′ viral genomic RNA (HO-ACCAGA-OH) inside the NCLP. (A) The Cryo-EM electrostatic potential of the 6 bases at the 5′ end of the viral RNA inside the NCLP (EM data base EMD-0142). The structures show gaps (asterisks) between bases 6 and 1, because the RNA (PDB 6H5S) molecules do not have a 5′ phosphate. (B) The protomer at the 3′ end inside the NCLP, showing that the last nucleotide is only bound to the base and not to the phosphate on the 2′ site on the ribose. The figure shows some of the residues binding the RNA, in red those that bind the bases. Reprinted with permission from Desfosses et al. (2019); Copyright 2019 National Academy of Sciences, used with permission. Frontier.

Figure 6
Figure 6

Base stacking over the interface between two protomers. The nucleoprotein binds 6 nucleotides, 3 point inward to the protein and 3 point outward. Often one cannot see clearly which base binds at the interface. (A) The structure of the RNA inside the NCLP of PIV5 (Alayyoubi et al., 2015). (B) The structure of the RNA inside the measles virus NCLP (Gutsche et al., 2015). (C) The structure of 6 nucleotides at the 5′ end of the viral RNA inside of the NCLP of measles virus (Desfosses et al., 2019). Because there is not a phosphate at the 5′, this shows the register of the RNA very clearly. The bases are shown, the phosphates are illustrated in red and the striped ovals show the stacking between two bases over the interface between two protomers.

Figure 7
Figure 7

Representation of N0P with N0 and the 304 residues of Ptail. (A) Localization of secondary structure and representation of flexibility/rigidity of Ptail with colors from red (flexible) to blue (rigid) and the transient α-helices from the N-terminal helix (α1 to α4). (B) Representation of N0P with the full Ptail showing α1 and α2 binding on N0, transient α3 and α4 region that weakly binds residues 96–127 of N0 as well the acidic loop between α3 and α4. For the N0 structure we used PDB 4CO6. Adapted from Milles et al. (2018); Copyright 2018 American Association for the Advancement of Science, used with permission. Frontiers.

Figure 8
Figure 8

Model of the polymerase binding to the 3′ end of the RNA inside the NC. (A) The polymerase (L) binds to the tetramerization domain of P and P binds to the nucleocapsid, at the linear helical motif of Ntail with the XD, and potentially also with α1 of P on the empty site on the last protomer of the nucleocapsid. In orange is the final 3′ end of the viral RNA. (B) The same as in (A) but now with the δα4 region on residues 96–127 of N0 that may take the polymerase closer to the 3′ end of the RNA. For this figure we used PDB 3ZDO for the tetramerization domain of P, PDB 1T6O for the structure of the complex between the linear helical motif of N and the XD of P, the structure of the polymerase of VSV with PDB 5A22 and the NCLP of measles virus with PDB 6H5S. Modified from Desfosses et al. (2019); Copyright 2019 National Academy of Sciences, used with permission.

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