Crystallographic Analysis of Rotavirus NSP2-RNA Complex Reveals Specific Recognition of 5' GG Sequence for RTPase Activity

Abstract

Rotavirus nonstructural protein NSP2, a functional octamer, is critical for the formation of viroplasms, which are exclusive sites for replication and packaging of the segmented double-stranded RNA (dsRNA) rotavirus genome. As a component of replication intermediates, NSP2 is also implicated in various replication-related activities. In addition to sequence-independent single-stranded RNA-binding and helix-destabilizing activities, NSP2 exhibits monomer-associated nucleoside and 5' RNA triphosphatase (NTPase/RTPase) activities that are mediated by a conserved H225 residue within a narrow enzymatic cleft. Lack of a 5' γ-phosphate is a common feature of the negative-strand RNA [(-)RNA] of the packaged dsRNA segments in rotavirus. Strikingly, all (-)RNAs (of group A rotaviruses) have a 5' GG dinucleotide sequence. As the only rotavirus protein with 5' RTPase activity, NSP2 is implicated in the removal of the γ-phosphate from the rotavirus (-)RNA. To understand how NSP2, despite its sequence-independent RNA-binding property, recognizes (-)RNA to hydrolyze the γ-phosphate within the catalytic cleft, we determined a crystal structure of NSP2 in complex with the 5' consensus sequence of minus-strand rotavirus RNA. Our studies show that the 5' GG of the bound oligoribonucleotide interacts extensively with highly conserved residues in the NSP2 enzymatic cleft. Although these residues provide GG-specific interactions, surface plasmon resonance studies suggest that the C-terminal helix and other basic residues outside the enzymatic cleft account for sequence-independent RNA binding of NSP2. A novel observation from our studies, which may have implications in viroplasm formation, is that the C-terminal helix of NSP2 exhibits two distinct conformations and engages in domain-swapping interactions, which result in the formation of NSP2 octamer chains.

Fig 1

Diagram schematic of a rotavirus dsRNA. The top schematic represents a dsRNA with the open reading frame (ORF) shown in gray, the 5′ and 3′ untranslated regions (UTRs) shown in orange, and the consensus sequences (CS) shown in red. The lower table shows the representative sequences of the 5′ and 3′ CS from different groups, strains, and gene segments of rotavirus. The conserved “GG” dinucleotide is shown in red.

Fig 2

Structure of NSP2-5′CS(−)RNA complex. (A) Simulated annealing composite omit map showing the 5′ terminal nucleotides in a (2|F_observed| − |F_calculated|) map calculated by using the protein only as the phasing model and contoured at ∼1.1 σ. Modeled RNA is shown in a ball-and-stick representation (yellow) inside the map, with the nitrogen and the oxygen atoms colored in blue and red, respectively. (B) Ribbon diagram representation of an NSP2 monomer in the “closed” conformation. (C) Ribbon diagram representation of an NSP2 monomer in the “open” conformation.

Fig 3

NSP2 octamer formation. (A) NSP2 tetramer in the crystallographic asymmetric unit as viewed along the pseudo 4-fold axis of the tetramer with bound RNA shown in a sphere representation. (B) NSP2 octamer formed by the tetramers related by the crystallographic 2-fold axis, as viewed along the pseudo 4-fold axis of the octamer. (C) NSP2 octamer viewed along the 2-fold axes showing the RNA-binding cleft. (D) Electrostatic surface potential surface of the NSP2 octamer (calculated without bound nucleotides) showing the groove (dashed black line) and two distinct electropositive patches: one near the cleft (white star) and the other at either end of the groove corresponding to the locations of the C-terminal helices in open (dashed black arrow) and closed (black arrow) conformations. Positively and negatively charged residues are shown in blue and red, respectively.

Fig 4

(A) Stereo view of interactions between NSP2 and 5′ CS of (−)RNA. The hydrogen bond interactions between the NSP2 residues (gray) and RNA are displayed as dashed lines. Residues involved in hydrophobic contacts are shown as spheres. A red dashed line indicates the cation-π interaction between R104 and G1. (B) Detailed interactions between SA11 NSP2 and RNA, as determined using LIGPLOT (36). The amino acid residues of NSP2 and the RNA involved in the interactions are labeled. Water molecules mediating RNA recognition are represented by small spheres in cyan. The carbon, nitrogen, and oxygen atoms are shown in black, blue, and red colored circles, respectively. Hydrogen bonds are shown as green lines between the respective donor and acceptor atoms, along with the acceptor-donor distance. The NSP2 residues involved in the hydrophobic interactions are indicated by an arc with spokes radiating toward the RNA atoms that they make contact with. The contacted atoms are shown with spokes radiating back.

Fig 5

Domain swapping of the C-terminal helix of NSP2. (A) Superposition of the NSP2 subunits with C-terminal helix in open (orange), and closed (blue) conformations. The dihedral angle changes in residues M293, K294, and P295 lead to the C-terminal helix switching its conformation. (B) Two NSP2 tetramers (green and red) interact with each other through swapped C-terminal helices. (C) Domain-swapping interactions of the C-terminal helices between the octamers lead to the formation of a chain of NSP2 octamers in the crystal lattice.

Fig 6

NSP2_ΔCT forms octamers. (A) Size-exclusion chromatography of wide-type and mutant NSP2s using Superose 6 (GE Healthcare), with elution volumes for each peak shown in the figure. (B) SDS-PAGE analysis of purified NSP2, NSP2_ΔCT, and NSP2_{K223A/R227A/ΔCT}. (C) Crystal structure NSP2_ΔCT octamer determined to 3.4-Å resolution (above) compared to the previously published NSP2 I422 octamer (11) (below). Two NSP2 subunits related by the 2-fold axis and involved in the tail-to-tail interaction of two NSP2 tetramers are shown in green and gold, respectively. The rest subunits of the octamers are shown in tan.

Fig 7

RNA-binding assays using SPR. (A) Representative sensograms for wild-type and mutant NSP2 at 50 nM (green), 100 nM (red), and 200 nM (black) binding to GG RNA. (B) Representative sensograms for wild-type and mutant NSP2 binding to CC RNA with the same color scheme shown in panel A. The association lasts 240 s and is followed by disassociation for 180 s. The deduced k_on, k_off, and apparent K_d values are shown in Table 2.