Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

Abstract

Paramyxoviruses and pneumoviruses infect cells through fusion (F) protein-mediated merger of the viral envelope with target membranes. Members of these families include a range of major human and animal pathogens, such as respiratory syncytial virus (RSV), measles virus (MeV), human parainfluenza viruses (HPIVs), and highly pathogenic Nipah virus (NiV). High-resolution F protein structures in both the metastable pre- and the postfusion conformation have been solved for several members of the families and a number of F-targeting entry inhibitors have progressed to advanced development or clinical testing. However, small-molecule RSV entry inhibitors have overall disappointed in clinical trials and viral resistance developed rapidly in experimental settings and patients, raising the question of whether the available structural information may provide a path to counteract viral escape through proactive inhibitor engineering. This article will summarize current mechanistic insight into F-mediated membrane fusion and examine the contribution of structural information to the development of small-molecule F inhibitors. Implications are outlined for future drug target selection and rational drug engineering strategies.

Keywords: Respiratory syncytial virus; antiviral therapeutic; entry inhibitor; measles virus; nipah virus; parainfluenzavirus; paramyxovirus; pneumovirus; virus entry.

Figure 1

The pneumo- and paramyxovirus entry machinery. (A) Schematic of paramyxovirus attachment and F proteins showing the overall head and stalk organization of the attachment protein tetramer and F protein trimer, respectively. (B) Color-coded schematic representation of the F protein domain organization (shown by example of RSV F). Heptad repeat (HR) domains A (HRA) and B (HRB) form the post- and prefusion F helical stalks, respectively. HR domain C (HRC) is located in the membrane-distal section of the prefusion F head. SP, signal peptide; FP, fusion peptide; TM, transmembrane domain; CT, cytoplasmic tail. The precursor F₀ precursor protein is cleaved into F₁ and F₂ subunits. (C) Crystal structure of the prefusion PIV5 F trimer (PDB 4GIP). Surface view with one monomer shown as cartoon (left panel). F₂ in blue and F₁ in salmon. Domain view of a single monomer (right panel), colored as in (B). (D) Postfusion hPIV3 F trimer (PDB 1ZTM), the 6HB is oriented towards the base of the structure. (E,F) Pre- and postfusion forms of RSV F (PDB 4MMQ and 3RRR, respectively). Subtle differences in overall geometry to paramyxovirus F are present in the prefusion head specifically. All figures were prepared with PyMol (DeLano Scientific; http://pymol.sourceforge.net/).

Figure 2

Prefusion pneumo- and paramyxovirus F structures complexed with small molecule inhibitors. (A) JNJ-53718678 (shown as yellow sphere) bound to RSV F (PDB 5KWW). The compound interacts with the base of the central F cavity and the fusion peptide, stabilizing the prefusion conformation. (B) Close-up of the hydrophobic binding pocket of JNJ-53718678 (yellow sticks) that involves all the three monomers of the F trimer. (C) MeV F in complex with small molecule entry inhibitor AS-48 (PDB 5YZC). The compound docks at the neck between F head and stalk, engaging residues in prefusion HR-B but not the fusion peptide. (D) Residue-view of the AS-48 (yellow sticks) target site.

Figure 3

Prefusion RSV F in complex with nAbs. (A) Docking poses of motavizumab (red and green) and AM14 (brown and smudge), bound to prefusion F. Both nAbs recognize quaternary epitopes (PDB 4ZYP) and bind the trimer alternatively (lower panel). (B,C) nAbs D25 and 5C4 (PDB 4JHW and 5W23, respectively) docked to the apex region of the prefusion RSV F protein trimer (antigenic site Ø). (D) Antigenic site Ø shown in orange and yellow. Mutation at position 201 mediates resistance.

Figure 4

Defined antigenic epitopes in pre- (left) and/or postfusion (right) RSV F. Shown are antigenic sites Ø and I-V. Sites Ø and V are prefusion F-specific, sites II-IV are present in both pre- and postfusion F, and site I is formed in postfusion F only. Ribbon representations of single RSV F monomers are shown for clarity, residues forming the individual epitopes are highlighted.

Figure 5

Co-crystal structures of prefusion NiV F with nAbs fab66 and 5B3. (A) Side and Table 66. bound to the apex region of the NiV prefusion F trimer (PDB 6T3F). Right panels show a close-up of the epitope, interacting residues are denoted as yellow sticks. (B) Side and top view of 5B3 bound to the lateral site of prefusion NiV F (PDB 6TYS). Two F monomers are engaged simultaneously in the interaction. Right panels: both heavy and light chains (depicted in green and brown, respectively) interact with the F protein.