Interactions between the Nucleoprotein and the Phosphoprotein of Pneumoviruses: Structural Insight for Rational Design of Antivirals

Abstract

Pneumoviruses include pathogenic human and animal viruses, the most known and studied being the human respiratory syncytial virus (hRSV) and the metapneumovirus (hMPV), which are the major cause of severe acute respiratory tract illness in young children worldwide, and main pathogens infecting elderly and immune-compromised people. The transcription and replication of these viruses take place in specific cytoplasmic inclusions called inclusion bodies (IBs). These activities depend on viral polymerase L, associated with its cofactor phosphoprotein P, for the recognition of the viral RNA genome encapsidated by the nucleoprotein N, forming the nucleocapsid (NC). The polymerase activities rely on diverse transient protein-protein interactions orchestrated by P playing the hub role. Among these interactions, P interacts with the NC to recruit L to the genome. The P protein also plays the role of chaperone to maintain the neosynthesized N monomeric and RNA-free (called N⁰) before specific encapsidation of the viral genome and antigenome. This review aims at giving an overview of recent structural information obtained for hRSV and hMPV P, N, and more specifically for P-NC and N⁰-P complexes that pave the way for the rational design of new antivirals against those viruses.

Keywords: HMPV; RSV; antivirals; nucleocapsid; nucleoprotein; phosphoprotein; pneumoviruses; protein-protein interaction; structure.

Figure 1

Pneumoviridae genomes and virion. (A) Genome organization of representative members of the Pneumoviridae family. Genomic RNAs are presented in sense (coding) orientation (3′-to-5′), with each box representing a gene encoding a separate mRNA drawn approximately to scale. The M2 gene encodes M1-2 and M2-2 proteins (represented by rectangles above M2 gene). (B) Representation of pneumovirus viral particle showing the structural proteins. Created with BioRender.com.

Figure 2

Schematic representation of the viral cycle of Pneumoviruses. Virion attachment to the cell is mediated by F and G proteins. The F protein is responsible for the fusion of viral and cell membranes, leading to the delivery in the cytoplasm of the NC complexed with L, P, and M2-1 proteins. Transcription and replication occur in membrane-less organelles called cytoplasmic inclusions bodies (IBs, light brown). Within IBs, M2-1 and viral mRNAs accumulate into sub-structures called inclusion body-associated granules (IBAGs, yellow). After viral protein production and genome replication, assembly and budding of new viral particles take place at the plasma membrane. Adapted from “Replication Cycle”, by BioRender.com (2020). Retrieved from https://app.biorender.com/biorender-templates (30 November 2021).

Figure 3

Schematic representation of the polymerase functioning of pneumoviruses. The polymerase L is responsible for both viral replication and transcription. The P protein plays a role in the hub by interacting with L and NC through its C-terminal P_CTD domain and with M2-1 and the monomeric and RNA-free N (N⁰) through its N-terminal P_NTD domain. Created with BioRender.com (30 November 2021).

Figure 4

Structure of Pneumovirus P proteins. (A) Domain architecture of hRSV and hMPV P proteins, with a fully disordered N-terminal domain, P_NTD, a short tetrameric coiled-coil oligomerization domain, P_OD (blue), and a C-terminal domain, P_CTD, consisting of a domain with a high α-helical propensity, P_Cα (red), and a highly disordered C-terminal tail, P_Ctail. The interaction regions of hRSV P with RdRp, or associated proteins like PP1, are indicated by arrows and bold lines. The corresponding regions in hMPV are also in bold lines. (B) High-resolution cryo-EM structures of the tetrameric L-associated hRSV and hMPV P proteins. Only the P_OD and P_Cα domains are observed in the L-P complex structures. Neither P_NTD nor P_Ctail, except for a single protomer, could be observed due to high disorder. Created with Pymol (https://pymol.org, 30 November 2021).

Figure 5

Structure of Pneumovirus N proteins. (A) Top (upper panel) and side (lower panel) views of hRSV N-RNA rings composed of 10 N proteins bound to RNA, purified from E. coli (PDB: 2WJ8 and 5FVC, respectively). N monomers are represented in ribbons; one N subunit is colored with the N_NTD in orange, N_CTD in yellow, and the N- and C-terminal parts in blue and green, respectively. The RNA is represented with the bases in black. N- and C-terminal extremities are indicated. (B) Left-handed N-RNA helix model (PDB: 4BKK). N monomers and RNA (black) atoms are shown as surfaces. One N subunit is colored with the N_NTD in orange, N_CTD in yellow, and the N- and C-terminal parts in blue and green, respectively. The C-terminal extremity of the N monomer at the top of the helix model is annotated. Created with UCSF ChimeraX [100].

Figure 6

X-ray structure of hRSV N protein protomer and of N_NTD in complex with P2 peptide and M76 compound. (A) An hRSV N protomer taken from the X-ray structure of N-RNA rings (PDB 2WJ8) is represented in ribbons, with the N_NTD in orange, N_CTD in yellow, and the N- and C-terminal arms in blue and green, respectively. The residues of N critical for the interaction with P are shown with lateral chains in black. N- and C-terminal extremities are indicated. (B) Zoom into the P2-binding site on N_NTD (left panel, P2 in cyan) (PDB: 4UCA) and of the M76 compound binding site (right panel, M76 in deep blue) (PDB: 4UCC). The residues of N involved in the interaction with P2 or M76 are indicated. Created with UCSF Chimera [104].

Figure 7

Structural changes involved in the transition from hMPV N⁰-P to oligomeric N-RNA forms. The transition requires the presence of RNA, as indicated at the bottom of the figure. (A) Crystal structure of hMPV N⁰- P_1-28 peptide complex (PDB: 5FVD). (B) Representation of a trimer of N in cartoon representation bound to RNA shown in black sticks (PDB: 5FVC). The black and red arrows symbolize the movement of the N C-arm and the release of P_1–28 peptide, respectively, required for N oligomerization. One N subunit is colored with the N_NTD in orange, N_CTD in yellow, and the N- and C-terminal parts in blue and green, respectively. The P_1-28 peptide is in pink. The N- and C-terminal extremities of N and the P peptide are indicated. Created with BioRender.com and UCSF ChimeraX [100].