Coupling of polymerase-nucleoprotein-RNA in an influenza virus mini ribonucleoprotein complex

Fig. 1. Overall structures of mini-vRNPs. The structures for the mini-vRNP in State-In and in State-Out forms are presented in (a, b), respectively. The representative 2D classifications are shown in the left panels. The cryo-EM densities for the mini-vRNPs are shown as isosurface rendering in the middle panels. The densities corresponding to PA, PB1, PB2, 3′ vRNA, or 5′ vRNA are colored cyan, pale cyan, light blue, red, and blue, respectively. NP-0 is colored in light yellow, while other NPs are displayed in white or light yellow. The models for the two states of the mini-vRNP are shown in the right panels as cartoon diagrams with the same color scheme as in the middle panels. The nucleotides bound in NP-0/+1/+2/+3 or in NP-1/-2/-3 are colored blue and red to indicate they are in the 5′ or the 3′ directions of vRNA, though they represent one single-stranded vRNA. Because the polypeptide of the PB2 C-terminal region (from residue 251 to the C-terminus) has only partially visible densities, the model of this part derived from a structure of influenza A virus polymerase (PDB code: 5M3H) is displayed as a semi-transparent cartoon.

Fig. 2. Interactions of FluPol and NP. a A side view of the structure of FluPol:NP-0:RNA is shown as a cartoon diagram with the same color scheme as used in Fig. 1. The body and head domains of NP-0 are indicated. The polypeptide of the C-terminal part of PB2 is displayed as a semi-transparent cartoon. b A close-up view of region 1 on the interface of FluPol and NP-0. Key residues and the nucleotides of 5′ vRNA are shown as colored sticks. The interactions surrounding NP-0R208 are enlarged and exhibited in the right panel. Key residues and the nucleotides are covered by cryo-EM densities as a gray mesh. Inter-molecular interactions are denoted as dashed lines. c A close-up view of region 2 on the interface of FluPol and NP-0. Key residues are shown as colored cartoons, and the side chains are shown as colored sticks. Key residues are covered by cryo-EM densities as gray mesh. Inter-molecular interactions are denoted as dashed lines. d Effect of PA and NP mutations on influenza A/Victoria/3/75 (H3N2) mini-vRNP activity. A neuraminidase (NA) vRNA template was used. Total RNA was isolated at 12 h post-transfection and analyzed by primer extension. WT, wild type; PA-3A, PA-S364A/K367A/N373A;…

Fig. 3. Interactions of RNA with FluPol and NPs. a Top view of the structure of FluPol:NP-0:RNA of the mini-vRNP in State-In is shown as a cartoon diagram with the same color scheme as used in Fig. 1. b The nucleotides bound in FluPol and NP-0 are shown as colored sticks and are covered by cryo-EM densities as colored mesh. An overall view is shown in the center of (b), and four close-up enlarged views are displayed in the frames. Several nucleotides are labeled to identify their positions. c An enlarged view of the D72-K90 loop region of NP-0 that inserts into the fork of the paired 5′/3′ vRNA. The residues R74, Y78, H82, and K87 of NP-0 are shown as colored sticks. The nucleotides at the fork of the double-stranded part of vRNA are labeled. d The structure of NP+1:NP+2:RNA unit. The polypeptides of NP+1 and NP+2 are presented as white and light yellow cartoons. The bound RNAs are shown as blue cartoons. The 3′ and 5′ directions of the bound RNA are indicated. e The nucleotides that bind to NP+2 are displayed as blue sticks and are covered by cryo-EM densities as blue mesh. Several nucleotides are labeled to identify their positions.

Fig. 4. Interactions of RNA with NP-0 and NP+2. The interaction of RNAs with NP-0 and NP+2 is analyzed in (a–f), respectively. a, d The structures of NP-0:RNA and NP+2:RNA are shown as cartoon diagrams with the same color scheme as in Fig. 1. b, e The molecules of NP-0 and NP+2 are depicted as molecular surface in white. The key residues of NP-0 that interact with 3′ RNA or 5′ RNA are colored as red and blue on the surface (b). The key residues of NP+2 that interact with RNA are colored as blue on the surface (e). The RNA molecules are shown as semi-transparent cartoons. Schematic of the protein-RNA interactions for NP-0:RNA (c) and NP+2:RNA (f). Solid and hollow circles indicate nucleotides that were visible or invisible, respectively. The amino acid residues interacting with each nucleotide (within 4.0 Å) are identified and listed next to their interacting nucleotides.