Measles virus proteins form liquid droplets where they can encapsidate their genomic material.

The processes of genome replication and transcription of SARS-CoV-2 represent important targets for viral inhibition. Betacoronaviral nucleoprotein (N) is a highly dynamic cofactor of the replication-transcription complex (RTC), whose function depends on an essential interaction with the amino-terminal ubiquitin-like domain of nsp3 (Ubl1). Here, we describe this complex (dissociation constant - 30 to 200 nM) at atomic resolution. The interaction implicates two linear motifs in the intrinsically disordered linker domain (N3), a hydrophobic helix ( ²¹⁹ LALLLLDRLNQL ²³⁰ ) and a disordered polar strand ( ²⁴³ GQTVTKKSAAEAS ²⁵⁵ ), that mutually engage to form a bipartite interaction, folding N3 around Ubl1. This results in substantial collapse in the dimensions of dimeric N, forming a highly compact molecular chaperone, that regulates binding to RNA, suggesting a key role of nsp3 in the association of N to the RTC. The identification of distinct linear motifs that mediate an important interaction between essential viral factors provides future targets for development of innovative strategies against COVID-19.

Measles virus genome encapsidation is essential for viral replication and is controlled by the intrinsically disordered phosphoprotein (P) maintaining the nucleoprotein in a monomeric form (N) before nucleocapsid assembly. All paramyxoviruses harbor highly disordered amino-terminal domains (P_NTD) that are hundreds of amino acids in length and whose function remains unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we describe the structure and dynamics of the 90-kDa N⁰P_NTD complex, comprising 450 disordered amino acids, at atomic resolution. NMR relaxation dispersion reveals the existence of an ultraweak N-interaction motif, hidden within the highly disordered P_NTD, that allows P_NTD to rapidly associate and dissociate from a specific site on N while tightly bound at the amino terminus, thereby hindering access to the surface of N. Mutation of this linear motif quenches the long-range dynamic coupling between the two interaction sites and completely abolishes viral transcription/replication in cell-based minigenome assays comprising integral viral replication machinery. This description transforms our understanding of intrinsic conformational disorder in paramyxoviral replication. The essential mechanism appears to be conserved across Paramyxoviridae, opening unique new perspectives for drug development against this family of pathogens.

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo‐proteins (N) that bind the entire genome. N‐RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self‐organization of N into NC‐like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real‐time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA‐sequence, with the genomic 5′ end and poly‐Adenine sequences assembling efficiently, while sequences such as poly‐Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process.

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5′) in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5′ and 3′ binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3′ end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.