Journal: Nature Communications

Article Title: Structural and functional analysis of the minimal orthomyxovirus-like polymerase of Tilapia Lake Virus from the highly diverged Amnoonviridae family

doi: 10.1038/s41467-023-44044-x

Figure Lengend Snippet: Summary of TiLV polymerase structures

Article Snippet: To trap TiLV polymerase in a late elongation, pre-termination state, 1.6 µM of TiLV polymerase were mixed with 1.3 µM of the TiLV vRNA 40-S loop (C23U) and 16 µM of capped RNA primer 13-mer in the cryo-EM buffer supplemented with 100 µM ATP, 100 µM UTP, 100 µM CTP, 100 µM GpCpp (Jena Bioscience) and 10 mM MgCl 2 .

Techniques:

Journal: Nature Communications

Article Title: Structural and functional analysis of the minimal orthomyxovirus-like polymerase of Tilapia Lake Virus from the highly diverged Amnoonviridae family

doi: 10.1038/s41467-023-44044-x

Figure Lengend Snippet: a TiLV polymerase in vRNA and cRNA pre-initiation state mode A. Top: Close-up of the 3′ vRNA end in the TiLV polymerase active site. The 3′ end is coloured in gold and nucleotides are numbered from the 3′ to the 5′, with G1 and G2 being in, respectively, the −1/+1 active site positions. PB1-C bridge domain, PB1-C thumb domain and PB2-N are in purple, aquamarine and dark red respectively. PB2-N/D102 from the lid domain is in spheres. Left: Schematic of the overall vRNA conformation with the +1 position within a dotted rectangle. Flexible nucleotides are in italics. Bottom: Close-up of the 3′ cRNA end in the TiLV polymerase active site, annotated as in a, except that C1 and G2 are, respectively, in the −1/ + 1 positions. Left: Schematic of the overall cRNA conformation annotated as in above. Nucleotide differences between the vRNA and the cRNA are highlighted in red. b TiLV polymerase in the vRNA initiation state with incoming CTP aligning with G2, annotated as in a. Magnesium ions (Mg 2+ ) are shown as green spheres. Left: Schematic of the overall RNA conformation, annotated as in a, with CTP at the +1 position within a dotted rectangle. c TiLV polymerase stalled in early elongation state with an internal 11 base pair (bp) product-template RNA duplex. Annotations as in a. The 3′ vRNA end (gold) is not yet pushed back by PB2-N D102 lid residue, shown as spheres. The 13-mer capped primer ending by…CC-3′ is in magenta and the incorporated nucleotides in cyan. A CpNHpp is in the +1 active site position. Magnesium ions (Mg 2+ ) are shown as green spheres. Right: Schematic of the overall RNA conformation with colours as above. d TiLV polymerase stalled in a late elongation, pre-termination state showing the canonical 10 base pair product-template RNA duplex, annotated as in a . After strand separation (enforced by PB2-N D102, shown as spheres) the template (gold) continues through the exit channel into the secondary 3′ end binding site. The 13-mer capped primer ending by…CC-3′ is no longer visible. Incorporated nucleotides are in cyan and GpCpp is in the +1 position. Right: Schematic of the overall RNA conformation with the v40-S C23U mutation highlighted in red. The GpCpp at the +1 position is within a dotted rectangle. Flexible nucleotides are in italics. e TiLV polymerase movements upon product elongation. The vRNA initiation state (grey) was superposed via PB1 with the early-elongation state (colours). The RNA is omitted for clarity. Only significant movements of PB1-C and PB2-N elements are shown (arrows). The PB2-N loop (24-32) in the vRNA initiation state, which has to displace to allow elongation, is surrounded by a dotted line.

Article Snippet: To trap TiLV polymerase in a late elongation, pre-termination state, 1.6 µM of TiLV polymerase were mixed with 1.3 µM of the TiLV vRNA 40-S loop (C23U) and 16 µM of capped RNA primer 13-mer in the cryo-EM buffer supplemented with 100 µM ATP, 100 µM UTP, 100 µM CTP, 100 µM GpCpp (Jena Bioscience) and 10 mM MgCl 2 .

Techniques: Residue, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Structural and functional analysis of the minimal orthomyxovirus-like polymerase of Tilapia Lake Virus from the highly diverged Amnoonviridae family

doi: 10.1038/s41467-023-44044-x

Figure Lengend Snippet: a Urea-PAGE analysis of in vitro TiLV polymerase activity using the 40-mer vRNA loop (v40) as template, without (lane 1, 3) or with (lane 2, 4) CpC dinucleotide, using ATP and UTP only (AU) (lane 1–2) or all NTPs (AUGC) (lane 3-4). Stalled product and putative full-length (FL) products are indicated on the right (post or pre-hook?). The decade marker (Nts) is on the left. Theoretical products are shown at the bottom of the gel. The 5′/3′ ends of the template are respectively coloured in plum and gold, the primer in magenta, the theoretically incorporated nucleotides in blue. Source data are provided as a Source Data file. (n = 6 independent experiments). b Urea-PAGE analysis of in vitro TiLV polymerase transcription-like activity using the v40 loop as template, ATP and UTP (AU) (lane 1-5) or all NTPs (AUGC) (lane 6-10), uncapped primers ending by…CC-3′ (lane 1, 6) or…CCA-3′ (lane 2, 7), capped primers ending by…CC-3′ of different lengths (11-mer, lane 3, 8; 12-mer, lane 4, 9; 13-mer, lane 5, 10). Expected stalled and full-length transcription-like products are indicated on the right. Read-throughs due to misincorporation are indicated with a star. Theoretical products are shown as in ( a ). Source data are provided as a Source Data file ( n = 6 independent experiments). c Urea-PAGE analysis of in vitro TiLV polymerase transcription-like activity using the v40-S loop with C23U mutation as template, ATP, UTP and CTP (AUC) (lane 1), CpC (lane 2), uncapped primers ending by…CC-3′ (lane 3) or…CCA-3′ (lane 4), capped primers ending by…CC-3′ of different lengths (11-mer, lane 5; 12-mer, lane 6; 13-mer, lane 7). Expected stalled products are indicated on the right. Read-throughs due to misincorporation are indicated with a star. Theoretical products are shown as in ( a ). The C23U mutation is coloured in red. Source data are provided as a Source Data file ( n = 3 independent experiments). d Urea-PAGE analysis of in vitro TiLV polymerase transcription-like activity using the v40-S loop with C23U mutation as template, all NTPs (AUCG) (lane 1), CpC (lane 2), uncapped primers ending by…CC-3′ (lane 3) or…CCA-3′ (lane 4), capped primers ending by…CC-3′ of different lengths (11-mer, lane 5; 12-mer, lane 6; 13-mer, lane 7). Expected stalled products are indicated on the right with. Read-throughs due to misincorporation are indicated with a star. Theoretical products are shown as in ( a ). Source data are provided as a Source Data file ( n = 3 independent experiments).

Article Snippet: To trap TiLV polymerase in a late elongation, pre-termination state, 1.6 µM of TiLV polymerase were mixed with 1.3 µM of the TiLV vRNA 40-S loop (C23U) and 16 µM of capped RNA primer 13-mer in the cryo-EM buffer supplemented with 100 µM ATP, 100 µM UTP, 100 µM CTP, 100 µM GpCpp (Jena Bioscience) and 10 mM MgCl 2 .

Techniques: In Vitro, Activity Assay, Marker, Mutagenesis