Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein

Abstract

The nucleoprotein (NP) of segmented negative-strand RNA viruses such as Orthomyxo-, Arena-, and Bunyaviruses coats the genomic viral RNA and together with the polymerase forms ribonucleoprotein particles (RNPs), which are both the template for replication and transcription and are packaged into new virions. Here we describe the crystal structure of La Crosse Orthobunyavirus NP both RNA free and a tetrameric form with single-stranded RNA bound. La Crosse Orthobunyavirus NP is a largely helical protein with a fold distinct from other bunyavirus genera NPs. It binds 11 RNA nucleotides in the positively charged groove between its two lobes, and hinged N- and C-terminal arms mediate oligomerization, allowing variable protein-protein interface geometry. Oligomerization and RNA binding are mediated by residues conserved in the Orthobunyavirus genus. In the twofold symmetric tetramer, 44 nucleotides bind in a closed ring with sharp bends at the NP-NP interfaces. The RNA is largely inaccessible within a continuous internal groove. Electron microscopy of RNPs released from virions shows them capable of forming a hierarchy of more or less compact irregular helical structures. We discuss how the planar, tetrameric NP-RNA structure might relate to a polar filament that upon supercoiling could be packaged into virions. This work gives insight into the RNA encapsidation and protection function of bunyavirus NP, but also highlights the need for dynamic rearrangements of the RNP to give the polymerase access to the template RNA.

Fig. 1.

Recombinant LACV NP characterization. (A) Gel filtration of NP tetramers purified from E. coli before and after treatment with RNase A and 0.4 M thiocyanate (discontinuous and continuous lines, respectively). Multiangle laser light scattering measurements for each peak are shown in dark and light gray lines, respectively. The mass observed before treatment is 131 KDa, corresponding to a tetramer with bound RNA. After treatment, there are two species of 126 KDa (tetramer with shorter RNA) and 26 KDa (monomer). (B) RNA content of tetramers purified from E. coli after various treatments. Lane 1, RNA isolated from tetramers directly purified from E. coli; lane 2, after treatment with RNase A; lane 3, after treatment with RNase A and 0.4 M thiocyanate. E. coli-derived RNAs of length 45–60 are preferentially bound to the tetramer. RNAs up to about 45 nts are protected from degradation, although there is some accessibility of RNase probably at the NP–NP interfaces. Thiocyanate destabilizes the tetramer, making more accessible the RNA that is not tightly bound to a single monomeric NP. (C) EM image of untreated tetramers negatively stained with 1% sodium silico-tungstate. The black line indicates 20 nm. Arrows point toward individual tetramers. (D) Electrophoresis mobility shift assay using native acrylamide gels of ³²P–labeled PH60 viral RNA with cold competitor RNA incubated for 1 h at room temperature with monomeric NP. Lane 1, labeled PH60 only; lane 2, labeled PH60 only incubated with monomeric N; lanes 3–6, labeled PH60 with 12 µM and successive threefold dilutions of cold PH60; lanes 7–10, the same with cold U-rich RNA.

Fig. 2.

Structure of the RNA-free LACV NP. (A) Ribbon representation of LACV NP with rainbow coloring from N- (blue) to C-terminus (red). Disordered hinge regions are dotted. Indicated secondary structure elements are consistent with the sequence alignment in Fig. 3. (B) Electrostatic surface of the LACV NP, the positive and negative charges, respectively, being in blue and red. (C) The N-arm oligomerization interface. The N-arm (blue sticks) forms a β-strand (βN) parallel with β2 on the body of the next protein (green with transparent surface). The three intermolecular main-chain hydrogen bonds are shown as yellow dotted lines. Asp8 and Arg40 form a salt-bridge. Residues Leu4, Phe6, Pro63, and Phe65 form a hydrophobic cluster. Asn45 side-chain makes two hydrogen bonds to the main-chain of Tyr7. (D) The C-arm oligomerization interface. Hydrophobic residues on one side of the C-arm amphipathic helix α11, notably Leu219, Ala223, Phe226, Leu227, Phe230, and Ile232 (red sticks), bind to a hydrophobic pocket on the body of the next protein (green ribbons and sticks with transparent surface) formed by residues from helices α7–α10 (notably Leu161, Ile165, Leu178, Leu182, Trp194, Met195, Ile202, and Leu206).

Fig. 3.

Sequence alignment of representative Orthobunyavirus NPs. The secondary structure of LACV NP is shown above and key functional residues colored as indicated.

Fig. 4.

RNA binding mode in tetrameric LACV NP. (A) Cartoon representation of LACV NP tetramer in complex with 44 nts RNA shown as cyan and orange sticks. A pseudo-twofold symmetry axis into the figure makes monomers A (dark blue) and C (light blue) equivalent as well as B (green) and D (lemon). (B) Correspondence between 2D projections of LACV NP–RNA tetramer crystal structure (Left, filtered to 20 Å) and EM class averages of LACV tetramers purified from E. coli and bound to bacterial RNA (Right). Class averages corresponding to top and tilted views (up to 30°) are displayed; side views are not observed. (C) RNA conformation within LACV NP–RNA tetramer showing S-shaped backbone conformation bound to each monomer and sharp kinks at the NP–NP interfaces. The top and bottom bends at the A–B and C–D interfaces are less sharp than the right and left bends at the D–A and B–C interfaces (Fig. S3A). Whereas the RNA backbone continuity across the NP–NP interfaces is clear between U11–U12, U22–U23, and U33–U34, the electron density for U43–U44 and the connectivity to U1 is less good, consistent with the fact that the RNA actually has 45 nts, one more than necessary to occupy all four NPs. (D–F) Orthogonal views of a surface representation of the NP–RNA tetrameric complex showing that the RNA is largely buried within interior grooves with only partial solvent access to mainly the backbone. The N- and C-arms are highlighted. (G) Schematic of protein–RNA interactions, highlighting the stacking arrangement of the bases. (H) Stick representation of NP–RNA interactions with the 11 nts RNA shown in cyan and interacting protein residues in yellow. Putative hydrogen bonds are shown as green dotted lines.

Fig. 5.

The hinged N- and C-arms enables flexible packaging of the genome. (A) Superimposition of the four monomers from the NP–RNA tetramer colored as in Fig. 4 showing the different positions of the N- and C-arms from the A/C and B/D pairs. (B) Superposition of LACV NP monomers from the RNA-free (red), tetrameric (chain A, blue and B, green), and P4₁ helical (orange) forms. The N- and C-arms of the helical form are respectively more similarly orientate to the B N-arm and A C-arm. The RNA-free arms (which result in an extended network in the crystal) are both distinct from other observed conformations. (C) Molecular packing of NP in a 4₁ helix as observed in the P4₁ crystal form, with the width and pitch of the helix indicated. RNA is modeled as an orange tube on the internal surface of the helix. Successive 11 nt RNA segments were positioned by superposing on the core of each helix protomer the B subunit from the tetramer together with its bound 11 nts of RNA. Without making any adjustments, the near continuity of the RNA segments shows that the RNA binding mode observed in the tetramer is compatible with a much longer RNA binding within an extended helix. (D) Near end view of 4₁ helical NP–RNA model showing resemblance of projection to the tetramer (Fig. 4A). (E) LACV RNPs from disrupted virions examined by electron microscopy after negative staining with 2% uranyl-acetate. The size of the RNP shown is ∼160 × 75 nm. The RNPs are flexible but in some areas display regions with apparent helical characteristics as highlighted in green and shown in close-up. (F and G) Purified LACV RNPs displaying partial (F) or complete (G) supercoiling. In G the RNP dimensions are ∼60 nm long × 18 nm wide. (H) RNPs are partially unwound after incubation at 37 °C and show long, thin necklaces representing individual NPs bound to RNA. A close-up view of three NPs bound to RNA with interprotein distance of ∼50 Å.