Insights into 5′ RNA hook coordination, distal duplex conformation, and 3′ template RNA sequestration.

(A) A top-down view of the 5′ vRNA hook binding site in the LASV L protein (PDB: 7OJN) [48] is shown. The L protein core is shown in grey, whereas the pyramid domain is shown in dark blue with the 5′ vRNA coloured yellow. Interactions between cognate base pairs are represented by dashed lines. A close-up on the LASV 5′ vRNA hook is shown with individual nucleotides labelled accordingly. For comparison, the 5′ RNA hook motifs of SFTSV (PDB: 8ASB) [50] and LACV (PDB: 7ORN) [49] are also shown. (B) A schematic depiction of the promoter structure at pre-initiation with the 5′ RNA in yellow, the 3′ RNA in purple, and the distal duplex region as well as the 5′ hook labelled. The 3′ RNA would proceed towards the RdRp active site. (C) The distal duplex of the SFTSV L early-elongation structure (PDB: 8ASB) [50] is shown demonstrating that the 5′ RNA (yellow) and 3′ RNA (purple) form a corkscrew-like motif that winds around the SFTSV vRBL. The 5′ RNA and 3′ RNA surface is shown at 50% transparency. A close-up of the key base pairs in the distal duplex region is provided revealing that the 5′ cRNA and 3′ cRNA are in fact shifted by 1 nucleotide allowing for more cognate base pair interactions to form. (D) A side-on view of the LACV vRBL in the late-elongation structure (PDB: 7ORI) [49] showing the 5′ vRNA bound to the LACV L hook binding site and the tail end of the 3′ vRNA bound to the 3′ secondary binding site is provided. As in (B), the 5′ RNA is coloured yellow, the 3′ RNA is coloured purple, and the RNA surface is shown at 50% transparency. A close-up of the 3′ vRNA in the 3′ secondary binding site is shown. Figures were created using CCP4mg [68]. LACV, La Crosse virus; LASV, Lassa virus; RdRp, RNA-dependent RNA polymerase; SFTSV, severe fever with thrombocytopenia syndrome virus; vRBL, vRNA-binding lobe; vRNA, viral RNA cRNA, complementary viral RNA.</p

Priming genome replication in the bunyaviruses.

(A) Analysis of the sequence conservation and nucleotide complementarity in the 5′ and 3′ terminal 20 nucleotides of the vRNA of all genome segments. Sequence conservation was analysed using Jalview [85]. Virus species in the following families were analysed: Arenaviridae (n = 14), Nairoviridae (n = 5), Hantaviridae (n = 14), Phenuiviridae (n = 14), and Peribunyaviridae (n = 18), sequences of all genome segments were used. (B) Structure-based model of initiation by prime-and-realign for genome replication exemplified for peribunyaviruses. The 5′ RNA is colored yellow and the hook shown as a line. The 3′ RNA is coloured purple with the 3-nt repeats colored light and dark purple. Incorporated nucleotides (product) are coloured in pink. The PR-loop is shown in dark blue except for the pre-initiation panel, where its position is speculative and it is therefore colored in grey. The binding pocket of nucleotides A6, C7, and A8 at priming stage, called buffer zone, is surrounded by a dotted square and labelled. The proposed successive steps (labelled above) of the models are presented from left to right. nt, nucleotide; PR-loop, prime-and-realign loop; vRNA, viral RNA.</p

Structural comparison of LASV, SFTSV, and LACV L proteins.

(A) The LASV late-elongation (PDB: 7OJN) [48], SFTSV late-elongation (PDB: 8ASD) [50], and LACV early-elongation (PDB: 7ORM) [49] L protein structures are shown side by side. The core of each L protein is shown in grey with key domains colored as follows: endonuclease (orange), pyramid domain (only in LASV, dark blue), CBD (cyan), mid-link domain (pink), and 627-like/zinc-binding domain (purple). Bound RNAs are shown as black ribbons. (B) The LASV late-elongation (PDB: 7OJN,) [48], SFTSV apo structure (PDB: 7ALP) [42], and LACV early-elongation (PDB: 7ORM) [49] structures are shown stacked in panels. Each panel contains a schematic linear representation of the L protein domain structure with the size of the individual domains scaled to represent their relative size in the full-length L protein. Individual domains are labelled and colored for clarity. The colors for each domain are mapped onto a cartoon representation of each L protein, which is then broken down into the influenza-corresponding PA-like, PB1-like, and PB2-like domains. Figures were created using CCP4mg [68] and Pymol (Schrödinger, LLC). CBD, cap-binding domain; CTER, C-terminal region; LACV, La Crosse virus; LASV, Lassa virus; SFTSV, severe fever with thrombocytopenia syndrome virus; vRBL, vRNA-binding lobe; vRNA, viral RNA; ZBD, zinc-binding domain ENDO, endonuclease domain.</p

Overview of bunyavirus L protein structures.

All published structures of bunyavirus L proteins are included in the table (JUNV, Junin virus; LACV, La Crosse virus; LASV, Lassa virus; MACV, Machupo virus; RVFV, Rift Valley fever virus; SFTSV, severe fever with thrombocytopenia syndrome virus). Some structures additionally include viral matrix protein Z (L+Z). Entries are organised firstly by year and then publication date. For each structure, the method of determination ( for X-ray crystallography, for cryo-EM) along with the calculated resolution, information on the protein conformational state (structures containing no viral RNA are marked as “apo”), accession codes for the Protein Data Bank (PDB) and/or Electron Microscopy Data Bank (EMDB) as well as the publication reference (Ref) are provided. The microscope and crystal emojis are open source from Microsoft (MIT license, https://github.com/microsoft/fluentui-emoji).</p

Comparison of bunyavirus L protein structures reflecting viral transcription.

The structural insights into bunyaviral transcription are limited to LACV L protein structures [49]. LACV L protein has been visualized during capped primer cleavage (PDB: 7ORJ), followed by the rotation of the ENDO for subsequent primer entry (PDB: 7ORK). In addition, the initiation (PDB: 7ORL) and early-elongation (PDB: 7ORM) steps could be visualized. Protein figures were generated using ChimeraX [92]. CBD, cap-binding domain; ENDO, endonuclease domain; LACV, La Crosse virus; ZBD, zinc-binding domain.</p

Comparison of bunyavirus L protein structures reflecting viral genome replication.

There are several structures of different bunyaviral L proteins that reflect different stages of viral genome replication. For SFTSV, early elongation (PDB: 8ASB), late elongation (PDB: 8ASD), and a resting state (PDB: 8ASG, Ref.) [50] have been visualized. For LASV, pre-initiation (PDB: 7OJL) and early elongation (PDB: 7OJN) [48] have been visualized. For LACV, pre-initiation (PDB: 6Z6G) [39], initiation (PDB: 7ORN), early elongation (PDB: 7ORO), and late elongation (PDB: 7ORI) [49] have been visualized. The RdRp active site is marked by a red dashed circle and labelled with A. Of note, we define late-stage elongation starting when the product-template duplex dissolves allowing the RNAs to exit the inner L protein cavity separately. Protein figures were generated using ChimeraX [92] and Pymol (Schrödinger LLC). CBD, cap-binding domain; ENDO, endonuclease domain; LACV, La Crosse virus; LASV, Lassa virus; PR-loop, prime-and-realign loop; RdRp, RNA-dependent RNA polymerase; SFTSV, severe fever with thrombocytopenia syndrome virus; ZBD, zinc-binding domain.</p

Structure of influenza virus polymerase complex for comparison.

The influenza virus polymerase complex (PDB: 4WSB) is presented as a schematic linear representation with the size of the individual domains scaled to represent their relative size in the trimeric polymerase complex. Individual domains are labelled and colored. The colors for each domain are mapped onto a cartoon representation of the trimeric complex, which is then broken down into the subunits PA, PB1, and PB2. Figures were created using Pymol (Schrödinger LLC). CBD, cap-binding domain; ENDO, endonuclease domain; NLS, nuclear localisation signal; PR-loop, prime-and-realign loop; vRBL, vRNA-binding lobe 627, 627-like domain, Cter, C terminus, Nter, N terminus.</p

RNA to Rule Them All: Critical Steps in Lassa Virus Ribonucleoparticle Assembly and Recruitment

Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming ribonucleoprotein complexes (RNPs) together with the viral RNA and the L protein. RNPs must be continuously restructured during viral genome replication and transcription. The Z protein is important for membrane recruitment of RNPs, viral particle assembly, and budding and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA, and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z, and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for the disassembly of ring-like NP trimers, a storage form, into monomers to subsequently form higher order RNA-bound NP assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of RNP assembly, recruitment, and release in Lassa virus

Presentation_1_Monomeric C-Reactive Protein in Serum With Markedly Elevated CRP Levels Shares Common Calcium-Dependent Ligand Binding Properties With an in vitro Dissociated Form of C-Reactive Protein.PPTX

A monomeric form of C-reactive protein (CRP) which precipitates with cell wall pneumococcal C polysaccharide (CWPS) and retains the ability to reversibly bind to its ligand phosphocholine has been produced through urea-induced dissociation at an optimized concentration of 3 M urea over a 10 weeks period. Dissociated samples were purified via size exclusion chromatography and characterized by western blot, phosphocholine affinity chromatography and CWPS precipitation. Human serum samples from patients with raised CRP levels (>100 mg/L as determined by the clinical laboratory assay) were purified by affinity and size exclusion chromatography and analyzed (n = 40) to determine whether circulating monomeric CRP could be detected ex vivo. All 40 samples tested positive for pentameric CRP via western blot and enzyme linked immunosorbent assay (ELISA) analysis. Monomeric C-reactive protein was also identified in all 40 patient samples tested, with an average level recorded of 1.03 mg/L (SE = ±0.11). Both the in vitro monomeric C-reactive protein and the human serum monomeric protein displayed a molecular weight of approximately 23 kDa, both were recognized by the same anti-CRP monoclonal antibody and both reversibly bound to phosphocholine in a calcium-dependent manner. In common with native pentameric CRP, the in vitro mCRP precipitated with CWPS. These overlapping characteristics suggest that a physiologically relevant, near-native monomeric CRP, which retains the structure and binding properties of native CRP subunits, has been produced through in vitro dissociation of pentameric CRP and also isolated from serum with markedly elevated CRP levels. This provides a clear route toward the in-depth study of the structure and function of physiological monomeric CRP.</p