Modes of paramyxovirus fusion: a Henipavirus perspective

Abstract

Henipavirus is a new genus of Paramyxoviridae that uses protein-based receptors (ephrinB2 and ephrinB3) for virus entry. Paramyxovirus entry requires the coordinated action of the fusion (F) and attachment viral envelope glycoproteins. Receptor binding to the attachment protein triggers F to undergo a conformational cascade that results in membrane fusion. The accumulation of structural and functional studies on many paramyxoviral fusion and attachment proteins, including the recent elucidation of structures of Nipah virus (NiV) and Hendra virus (HeV) G glycoproteins bound and unbound to cognate ephrinB receptors, indicate that henipavirus entry and fusion could differ mechanistically from paramyxoviruses that use glycan-based receptors.

Figure 1. Relative positions of the ligand binding sites and dimer interfaces of the Hendra virus (HeV-G), Measles virus (MeV-H) and Parainfluenza virus 3 (PIV3-HN) attachment glycoproteins

(a) Cartoon representations of HeV-G, MV-H and PIV3-HN (PDB accession no. 2X9M, 3INB, and 1V2N, respectively) in their dimeric form are shown with the left monomer in each case anchored in the same orientation. The central axis of the six-bladed beta-propeller in each left monomer is pointing towards the reader, and each blade is color coded as indicated (b1;red, b2;blue, b3;green, b4;cyan, b5;gray, b6;magenta). (b) Surface representation of the cartoon structures are shown. The ligand binding residues are highlighted in green (ephrinB2 on HeV-G), red (CD46 on MV-H), and magenta (sialic acid on PIV3-HN). Residues involved in monomer-monomer interactions at the dimer interface are shown in blue and may not be fully visible in this view. According to the analysis performed by Bowden et al[21], relative to the angle of association between HeV-G dimers (set at 0°), the angle of association between MeV-H and PIV3-HN dimers is 63° and 31°, respectively. In other words, if the left monomer for all three attachment proteins is anchored to match the orientation of HeV-G, then the right monomer of MV-H would have to rotate 63° away from the reader into the page in order for it to line up with the right monomer of HeV-G. In contrast, the right monomer of PIV3-HN would have to rotate 31° towards the reader in order for the same alignment to be achieved. (c) Dimers in panel B are rotated 90° towards the reader so that the ephrinB2 binding site on HeV-G is now facing upwards towards the direction of the target cell. (d) To better visualize the dimer interacting surface the monomers in each dimer are separated with a motion akin to opening a pair of French windows from the outside. That is, each monomer is rotated 90° towards the reader. The dimer interacting residues colored in blue are now more easily visualized and the approximate area buried in the dimer interfaces are circled. The dimer-interface areas were calculated using PISA as 876.2 Ų, 1075 Ų, 1780.2 Ų for HeV-G, MV-H and PIV3-HN, respectively.

Figure 2. Schematic representation of the Nipah virus fusion (NiV-F) protein and major structural transitions during the fusion cascade

(a) Linear schematic of NiV-F. Domains I, II, and III are colored cyan, brown, and purple, respectively. The fusion peptide (FP) is a stretch of ~25 residues (magenta box) immediately C-terminal of the F cleavage site (upright arrow). Cathepsin L cleaves F into disulfide linked F₁ and F₂ subunits. The N- (HR1) and C- (HR2) terminal heptad repeats are both in the F₁ subunit. HR1 is located in the DIII domain and the HR2 is close to the transmembrane region. A third heptad repeat (HR3) is found in the F₂ subunit, and forms part of the DIII core that helps to stabilize the HR1 segments in the pre-fusion form. The relative positions of the 4 utilized N-linked glycosylation sites (F2–F5) are indicated by branched stick figures. Schematic of the pre-fusion (b), pre-hairpin intermediate (c), and postfusion form of F (d) adapted from Russell et al [57]. The color scheme for the major structural features are as indicated in (a). In the pre-fusion form (b), the individual segments of HR1 (yellow and green) are in a variety of secondary structures tucked into stable elements of the DIII core at the top of the trimer. For clarity, the boxed insert shows an isolated HR1 region undergoing F-triggering. Receptor induced allosteric signals from the attachment protein induces release of the fusion peptide (FP, magenta), which then inserts itself into the host cell membrane. FP insertion facilitates re-folding of the disparate HR1 segments into an extended alpha helix. The fusion cascade now progresses through a transitional form known as the pre-hairpin intermediate (PHI) shown in (c). Re-folding of the HR1 segments forms an N-terminal trimeric coiled coil that now anchors F to the host cell membrane. The HR2 alpha-helices (orange) that form the stalk of the pre-fusion structure disassemble, and translocate 180° to pack against the interstices of the HR1 trimer in an anti-parallel fashion. Packing of the HR2 segments onto the trimeric HR1 core forms a six-helix bundle (6-HB) structure, which necessarily brings the viral membrane in close apposition to the host cell membrane (not shown). Formation of the stable 6-HB structure drives merger of the virus and cell membranes that results in membrane fusion. The post-fusion structure of F with the 6-HB is shown in (d).

Figure 3. Structural models for the pre- and post- fusion forms of Nipah virus F

Models were generated using 3D-PSSM (http://www.sbg.bio.ic.ac.uk) based on the pre- and post-fusion PIV5-F [56] and hPIV3-F [9] structures, respectively (a) Trimeric model of the pre-fusion form of NiV-F. HR1 and HR2 sequences that eventually fold into the six-helix bundle in the post-fusion form are colored in yellow and orange, respectively. The F3 (Asn 99) and F5 (Asn 464) N-glycan sites are indicated by blue (F3) and red (F5) ball and stick figures. To better appreciate the location of the F3 and F5 N-glycans with respect to conserved features of the trimer (HR1 and HR2 regions), the trimer is rotated 90° towards the reader such that the reader is looking down the three-fold axis of the F protein from the perspective of the target cell membrane. This top-on view is shown at three different z levels represented by the dotted lines. From the top of the molecule (z₀ view), the labeled F3 N-glycan site can be seen in close proximity to the cleavage site (asterisk) and the fusion peptide loop (colored black). The HR1 region (yellow) is nicely tucked into a cradle at the vertices of the trimer. For clarity, portions above the dotted lines (distal to viral membrane) at the z₁ and z₂ levels have been removed. (b) Trimeric model of the post-fusion form where the head, neck, and stalk domains are indicated. (c) The six-helix-bundle (6-HB) is shown from the perspective of the central three-fold axis just N-terminal (membrane distal) to the HR-2 region. For comparisons, the monomer of both the pre and post-fusion form are shown in (d) and (e), respectively. The HR1 and HR2 regions and the F3 and F5 N-glycan sites are colored as in the pre-fusion structure.