Self-capping of nucleoprotein filaments protects the Newcastle disease virus genome

Abstract

Non-segmented negative-strand RNA viruses, such as measles, ebola and Newcastle disease viruses (NDV), encapsidate viral genomic RNAs into helical nucleocapsids, which serve as the template for viral replication and transcription. Here, the clam-shaped nucleocapsid structure, where the NDV viral genome is sequestered, was determined at 4.8 Å resolution by cryo-electron microscopy. The clam-shaped structure is composed of two single-turn spirals packed in a back-to-back mode. This tightly packed structure functions as a seed for the assembly of a nucleocapsid from both directions, facilitating the growth of double-headed filaments with two separate RNA strings inside. Disruption of this structure by mutations in its loop interface yielded a single-headed unfunctional filament.

CryoEM structure of the Nipah virus nucleocapsid assembly

“…Structures of nucleocapsid-like assemblies of several paramyxoviruses, assembled as helical [12], ring [13] or clam-shaped [14] complexes, have been already reported. However, the nucleocapsid of NiV shares only 32% sequence identity with the nucleocapsid of the Measles virus, the closest homologue with an available structure.…”

CryoEM structure of the Nipah virus nucleocapsid assembly

“…The encapsidated genome serves as a template for both mRNA transcription and viral genome replication. Structural analysis of a spiral assembly, formed by a fulllength recombinant NiV N protein, reported here (Figure 1), revealed an architecture common to other RNA-bound Paramyxoviridae family N proteins 9,11,14 , indicating that the overall mechanism for encapsidation of the viral genome is universal among members of this family. Upon binding to the RNA, the monomeric NiV N protein undergoes a conformational change involving a 28 rotation via the hinge motion between the two Ncore domains, effectively closing the two lobes around the RNA resulting in tight binding.…”

“…Interactions across this interface are mediated by hydrogen bonding and polar interactions made by loop segments A1-H2, A2-H5 and H6-H7 ( Figure 4B) from each opposing protomer. 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 9 . This is due to a significantly tighter interaction between the halves of the NiV clam shell as compared to the NDV assembly.…”

“…Interestingly, two distinct types of clam-shaped assemblies are observed for the NiV N protein: (i) a spiral clam-shaped conformation, which is formed by two spirals stacking against each other; and (ii) a semi-spiral clam-shaped assembly, formed by the stacking of ring-like and spiral oligomers. Previous mutational analysis, in NDV and SeV, of residues corresponding to the A2-H5 loop ( Figure 4B), which facilitates formation of the clam-shaped assembly, has resulted in up to 100% loss of in vitro replication while retaining the helical assembly capability of the N protein 9,17 . However, the molecular mechanism underlying this loss of activity is unknown, since the RdRp synthesizes the new RNA strand by initiating at the promoter encoded in the 3ʹ end of RNA template 18 , and hence is not expected to interact with the clam-shaped assembly that accommodates the 5ʹ end of the RNA template.…”

“…Aside from the common spiral assembly, about 35 % of N protein particles were found as clam-shaped assemblies which can be further classified into two primary distinct conformations, a spiral clam-shaped assembly and a semispiral clam-shaped assembly. The spiral clam-shaped assembly is composed of two N protein spirals stacked face to face, as seen for the Newcastle Diseases virus (NDV) N protein assembly 9 . In contrast, the semi-spiral clam-shaped assembly features one 14 protomer N protein ring and one N protein spiral stacked together ( Figure S3).…”