Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (N_Δ21) and a peptide of 60 amino acids (P₆₀) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N⁰). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis. The negative sense RNA genome of the rhabdoviruses is encapsidated by the nucleoprotein, and the replication of the genome requires a continuous supply of RNA-free, monomeric nucleoprotein (N⁰) to encapsidate the newly synthesized (+)RNA intermediate antigenomes and (−)RNA genomes. In this process, the viral phosphoprotein acts as a chaperone, forming a heterodimeric complex, named N⁰-P, which prevents nascent N molecules from self-assembling and from binding to cellular RNAs. We reconstructed the N⁰-P complex of the prototype rhabdovirus, vesicular stomatitis virus, and characterized its structure by crystal X-ray diffraction and solution experiments. Our results show how the N-terminal region of the phosphoprotein folds upon binding to the RNA-free nucleoprotein and how it prevents the non-specific encapsidation of host-cell RNA. This complex is soluble and heterodimeric, but by forcing it to polymerize into a crystal it associated into a circular decamer of heterodimers very similar to the previously crystallized decameric N-RNA ring. On the basis of our results, we propose a model that explains the role of the phosphoprotein in the encapsidation of newly synthesized RNA and in the initiation of RNA synthesis by the viral polymerase.

Significance: Thymosin-β4 (Tβ4) sequesters actin monomers to help maintain the high concentrations of unpolymerized actin in higher eukaryotic cells. Despite more than two decades of research investigating the Tβ4–actin interaction, the X-ray structure of the full-length Tβ4:actin complex remained unresolved. Here, we report two X-ray structures of Tβ4:actin complexes. The first structure reveals that Tβ4 has two helices that bind at the barbed and pointed faces of actin, whereas the second structure displays a more open actin nucleotide binding cleft and a disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, reveal how Tβ4 prevents monomeric actin from joining actin filaments but participates in the exchange of actin with profilin to ensure controlled actin polymerization.

Significance: The surface of mammalian cells contains abundant plasma membrane invaginations termed “caveolae.” Caveolae are important for various cellular functions, e.g. signaling, membrane regulation, and vesicular trafficking. Assembly and stability of caveolae depends on a protein coat formed by complexes of cavin and caveolin proteins whose structure has remained elusive. To understand the architecture of caveolae, we structurally analyzed cavin and caveolin complexes and visualized caveolae in cells by electron cryomicroscopy. A regular polyhedron emerged as the most likely architectural principle of caveolae. Polyhedra provide the underlying principle in many biological structures in which spherical curvature is imposed by proteins. We suggest a model for the caveolar coat architecture based on regularly arranged, net-like cavin assemblies and disc-shaped caveolin oligomers.

Non-segmented, (-)RNA viruses cause serious human diseases. Human metapneumovirus (HMPV), an emerging pathogen of this order of viruses (Mononegavirales) is one of the main causes of respiratory tract illness in children. To help elucidate the assembly mechanism of the nucleocapsid (the viral RNA genome packaged by the nucleoprotein N) we present crystallographic structures of HMPV N in its assembled RNA-bound state and in a monomeric state, bound to the polymerase cofactor P. Our structures reveal molecular details of how P inhibits the self-assembly of N and how N transitions between the RNA-free and RNA-bound conformational state. Notably, we observe a role for the C-terminal extension of N in directly preventing premature uptake of RNA by folding into the RNA-binding cleft. Our structures suggest a common mechanism of how the growth of the nucleocapsid is orchestrated, and highlight an interaction site representing an important target for antivirals.