Structure and assembly of double-headed Sendai virus nucleocapsids

Abstract

Paramyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are encapsidated by nucleoproteins into helical nucleocapsids. Here, we reported a double-headed SeV nucleocapsid assembled in a tail-to-tail manner, and resolved its helical stems and clam-shaped joint at the respective resolutions of 2.9 and 3.9 Å, via cryo-electron microscopy. Our structures offer important insights into the mechanism of the helical polymerization, in particular via an unnoticed exchange of a N-terminal hole formed by three loops of nucleoproteins, and unveil the clam-shaped joint in a hyper-closed state for nucleocapsid dimerization. Direct visualization of the loop from the disordered C-terminal tail provides structural evidence that C-terminal tail is correlated to the curvature of nucleocapsid and links nucleocapsid condensation and genome replication and transcription with different assembly forms.

CryoEM structure of the Nipah virus nucleocapsid assembly

“…The surface area, buried at the clam-shell interface of each monomer, is about five times larger than seen in the clam-shaped assembly of NDV, where only one protein loop (residues 104-124) is involved in the interaction [14]. This likely creates a closer interaction between the halves of the NiV clam shell as compared to the NDV assembly, similar to that observed in the recently reported clam-shaped assembly of the Sendai virus (SeV) [42]. Interestingly, although sequences of these clam-shell interface loops are not conserved between NiV, NDV and SeV, or other members of the Paramyxoviridae, these loops are rich in glycine and proline which can facilitate conformational flexibility (Fig 5C).…”

“…Similarly, these assemblies may also lead to the budding of virions which contain multiple RdRp-nucleocapsid assemblies [59]. Similar clam-shaped assemblies were observed for NDV and most recently for the Sendai virus (SeV) (S9 Fig), both for recombinantly produced protein and for nucleocapsids purified from virions, and it has been proposed they may act as a seed for formation of a double headed spiral assembly [14,42], although the possibility cannot be excluded that these NDV and SeV assemblies may have formed from damaged fragments of virionic nucleocapsids [60]. Nevertheless, as NiV N shares only 28% and 29% sequence identity with the NDV and SeV nucleocapsids,…”

Structural plasticity of mumps virus nucleocapsids with cryo-EM structures

“…Therefore, the N-hole adopts the same domain swapping process as the N-arm and contributes to the assembly of MuV N ring-13p and N ring-14p . Recently, similar N-hole-like motifs have also been reported to stabilize SeV nucleocapsids 31 .…”

Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions

“…This may reflect either the weak secondary structural preferences indicated by NMR chemical shift measurements (Figure S4), and/or the chainstiffening effects of the six proline residues that are distributed throughout the linker. The contour length of the linker (192 Å) clearly allows for multivalent attachment of the binding domains of the P protein to the nucleocapsid (the inter-subunit distances within the nucleocapsid being of the order of 50 Å [9][10][11]13,14]). However, it is presently unknown if this is required for polymerase function.…”

“…All paramyxoviruses require three proteins to facilitate virally-directed RNA synthesis; one protein to package, protect and organize the RNA genome, and two proteins that together form the viral RNA-dependent RNA polymerase (RdRp). The genome is packaged by the nucleocapsid protein (N protein), resulting in formation of a helical protein-RNA complex termed the nucleocapsid [9][10][11][12][13][14]. The viral RdRp [15][16][17][18] performs transcription and genome replication sequentially, using the nucleocapsid as a template [19].…”