Functional transcriptional regulatory sequence (TRS) RNA binding and helix destabilizing determinants of murine hepatitis virus (MHV) nucleocapsid (N) protein

Abstract

Coronavirus (CoV) nucleocapsid (N) protein contains two structurally independent RNA binding domains. These are denoted N-terminal domain (NTD) and C-terminal domain and are joined by a charged linker region rich in serine and arginine residues (SR linker). In mouse hepatitis virus (MHV), the NTD binds the transcriptional regulatory sequence (TRS) RNA, a conserved hexanucleotide sequence required for subgenomic RNA synthesis. The NTD is also capable of disrupting a short RNA duplex. We show here that three residues on the β3 (Arg-125 and Tyr-127) and β5 (Tyr-190) strands play key roles in TRS RNA binding and helix destabilization with Ala substitutions of these residues lethal to the virus. NMR studies of the MHV NTD·TRS complex revealed that this region defines a major RNA binding interface in MHV with site-directed spin labeling studies consistent with a model in which the adenosine-rich 3'-region of TRS is anchored by Arg-125, Tyr-127, and Tyr-190 in a way that is critical for efficient subgenomic RNA synthesis in MHV. Characterization of CoV N NTDs from infectious bronchitis virus and from severe acute respiratory syndrome CoV revealed that, although detailed NTD-TRS determinants are distinct from those of MHV NTD, rapid helix destabilization activity of CoV N NTDs is most strongly correlated with CoV function and virus viability.

FIGURE 1.

Schematic of MHV N protein domain organization. Linker regions are depicted as black lines, and regions of defined secondary structure are shown as rectangles. M, membrane protein; CTD, C-terminal domain.

FIGURE 2.

MHV NTD structure and RNA binding assay. A, ribbon diagram of the crystal structure of MHV NTD (Protein Data Bank code 3HD4) (18). Putative RNA binding residues are drawn as sticks and colored yellow (Thr-73, orange). B, fluorescence anisotropy titrations of NTD mutants to 5′-F-labeled TRS in 50 mm KP_i, pH = 6.0, 100 mm KCl at 25.0 °C. Solid lines represent the best fit to a single site binding model (supplemental Methods, fitting model 1) with parameters compiled in Table 1. N(1–230), open circles (○); N(60–230), open up triangles (△); N219 Y105A, open squares (□); N219 H107A, solid up triangles (▴); N219 R125A, solid circles (●); N219 R125A/T73K, solid down triangles (▾); N219 H136A, open diamonds (◊); N219 Y190A, solid squares (■).

FIGURE 3.

TRS RNA binds in defined strand polarity on NTD. A, chemical shift perturbations of the N197·TRS complex at 600 MHz. Chemical shift perturbations are painted on the crystal structure of the free protein, colored from yellow to red (small to large chemical shift changes). B, line broadening that results from N197 binding to TRS RNA measured at 800 MHz. The log of peak intensity is painted on the crystal structure of the free protein, colored from blue to yellow (more broadening to less broadening). C, additional line broadening that results from incubation with the 5′-AU*CUAAACUU-3′ TRS RNA in complex with N197 acquired at 800 MHz. The crystal structure of the free protein is colored as described in B. Regions of the protein that are differentially broadened in the paramagnetic versus diamagnetic spin label RNA are colored in red. D, additional line broadening that results from incubation with 5′-AUCUAAACU*U-3′ TRS RNA in complex with N197 acquired at 800 MHz. The crystal structure of the free protein is colored as described in B. Regions of the protein that are differentially broadened due to the effect of the paramagnetic spin label are colored in red. The right-hand view is an approximate 45° rotation along the z axis of the left-hand view.

FIGURE 4.

N219 mutants destabilize TRS-cTRS duplexes. A, general schematic of the FRET-based assay for monitoring TRS-cTRS duplex destabilization for N219 mutants that fully dissociate the TRS-cTRS duplex into component single strands (E_FRET = 0). hv, excitation at 520 nm. B, plot of FRET efficiency as a function of total NTD concentration using 50 nm 5′-Cy3-TRS and 3′-Cy5-cTRS RNAs in 50 mm KP_i, pH = 6.0, 100 mm KCl at 25.0 °C. N(1–230), open circles (○); N(60–230), open up triangles (△); N219 Y105A, open squares (□); N210 H107A, solid up triangles (▴); N219 R125A, solid circles (●); N219 H136A, open diamonds (◊); N219 Y190A, solid squares (■). The solid lines represent the best fit to a model that assumes the NTD forms a 1:1 complex with the single strands with the affinities fixed to their known values (see Table 1) and the affinity for the duplex, K₄, optimized in the fit (supplemental Methods, fitting model 2). In all cases, K₄ ≪ 1 (see also Ref. 20), which is representative of negligible affinity of the NTDs for the duplex. C, kinetics of strand destabilization at saturating concentrations (15 μm) of N219 mutants. N219 R125A, solid circles (●); N219 Y190A, solid squares (■). The solid line represents the best fit to a single exponential decay with parameters compiled in Table 1.

FIGURE 5.

Arg-125 and Tyr-190 are required for subgenomic RNA synthesis by MHV. A, plaque size of viruses containing mutations in the N gene. The bar graph represents the mean and error bars represent the S.D. of 40 well formed plaques. B, number of infectious centers that result from electroporation of BHK-R cells with WT MHV genome and an N helper gene represented as the mean and error bars represent the S.D. from three experiments.

FIGURE 6.

Global structural overlay of CoV N protein NTDs. A, IBV NTD (gray) (Protein Data Bank code 2BTL (22)) and MHV NTD (slate) (Protein Data Bank code 3HD4 (18)) align with an ∼1.3-Å root mean square deviation (for 89 C-α atoms). Residues that are critical for MHV NTD/MHV TRS interactions are shown as sticks and colored yellow. Analogous residues in IBV are shown as sticks and colored gray. Residues that have been shown to be critical for infectivity in IBV, Arg-76 and Tyr-94 (36), are shown as sticks and colored orange. Analogous residues in MHV, Arg-110 and Tyr-129, that have no impact on infectivity (18) are shown as sticks and colored magenta. B, SARS-CoV NTD (light blue) (Protein Data Bank code 2OFZ (37)) and MHV NTD (slate) (Protein Data Bank code 3HD4 (18)) align with a root mean square deviation of 0.87 Å (for 89 C-α atoms). As in A, residues critical for TRS binding in MHV are shown as sticks in yellow. Analogous residues in SARS-CoV NTD are shown as sticks and colored cyan.

FIGURE 7.

Helix destabilization is conserved characteristic of CoV N protein NTDs. Saturating concentrations of IBV NTD WT (15 μm; blue solid circles (●)), IBV NTD R76A (25 μm; open circles (○)), IBV NTD Y94A (25 μm; cyan solid squares (■)), and SARS NTD WT (10 μm; red solid up triangles (▴)) were titrated into a TRS-cTRS duplex while monitoring the FRET efficiency as a function of time (left). After E stabilized for each protein, saturating (1 μm) MHV NTD was added (middle). The bar graph represents the average of E over ∼600 s with the S.D. displayed as error bars. The protein was then dissociated from the RNA by addition of 300 mm KCl (right). The bar graph represents that average of E over ∼500 s with the standard deviation displayed as error bars.

FIGURE 8.

Schematic model for NTD·TRS interactions. TRS binds across the β-platform of NTD (see Fig. 2A) in a defined strand polarity determined by a nitroxide radical spin labeling experiment with the 5′-end of TRS near β4 and the 3′-end near β5. Arg-125, Tyr-127, and Tyr-190 make important contacts with TRS, likely anchoring the -AAA- motif. Only secondary structural elements of NTD are schematized for clarity with the amino acids involved in the TRS interaction highlighted on the core of the protein. Locations of 4-thiouridine incorporation are marked with an asterisk.