Recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein forms a dimer through its C-terminal domain

Abstract

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The viral nucleocapsid (N) protein plays an essential role in viral RNA packaging. In this study, recombinant SARS-CoV N protein was shown to be dimeric by analytical ultracentrifugation, size exclusion chromatography coupled with light scattering, and chemical cross-linking. Dimeric N proteins self-associate into tetramers and higher molecular weight oligomers at high concentrations. The dimerization domain of N was mapped through studies of the oligomeric states of several truncated mutants. Although mutants consisting of residues 1-210 and 1-284 fold as monomers, constructs consisting of residues 211-422 and 285-422 efficiently form dimers. When in excess, the truncated construct 285-422 inhibits the homodimerization of full-length N protein by forming a heterodimer with the full-length N protein. These results suggest that the N protein oligomerization involves the C-terminal residues 285-422, and this region is a good target for mutagenic studies to disrupt N protein self-association and virion assembly.

Fig. 1

Expression and purification of recombinant SARS-CoV N protein.A, SDS-PAGE. Lanes are as follows: lane 1, protein marker; lane 2, lysate from uninduced cells; lane 3, lysate from cells induced by 1 mm isopropyl-1-thio-β-d-galactopyranoside for 4 h; lane 4, supernatant of the induced lysate centrifuged at 75,000 × g for 30 min; lane 5, elution from the first cation exchange column (SP-Sepharose); lane 6, elution from the second cation exchange column (Source 15S); lane 7, elution from size exclusion column (Superdex 200). The arrow points to the band corresponding to the N protein. B, fast protein liquid chromatography gel filtration chromatogram of purified N protein. The column (Superdex 200 16/60) was calibrated with a mixture of standard proteins with molecular masses as shown. AU, absorbance units.

Fig. 2

Nonspecific binding of SARS-CoV N protein with (A) yeast tRNA and (B) 54-mer ssDNA. N proteins were kept at 10 μm final concentration, and oligonucleotides were added at increasing nucleic acid:protein molar ratios. The reaction mixtures were electrophoresed on native 0.8% agarose gels. The first lanes of each gel contain protein only. The gels on the left are stained with Coomassie Blue for detection of protein, and the gels on the right are stained with ethidium bromide for detection of nucleic acid.

Fig. 3

Oligomerization of the recombinant N protein.A, estimation of the native molecular mass by SEC-LS. The solid line corresponds to UV trace of the protein eluting from Superose 6 column, and the dotted line represents the average molecular weight calculated by ASTRA software at 5-μl intervals. B, sedimentation coefficient distributions calculated for N protein from sedimentation velocity studies. Shown are plots of the distribution of molecules with a given s value against the sedimentation coefficient (s) for proteins at 1 (solid line) and 25 μm (dotted line). These distributions are described by a corrected weight average sedimentation coefficient $S_{20,\text{water}}^0=3.9$ S and a calculated frictional ratio (f/f_o) of 2.0. C, the effect of protein concentration on the equilibrium distribution of N protein from sedimentation equilibrium studies. Shown are plots of protein concentration in fringes against radius²/2 cm² at three concentrations. Only 1 in 30 data points are shown, although all were used in the analysis. The lines are calculated for an ideal distribution that includes a dimer/tetramer equilibrium. The calculated mass for the dimer is 92.4 kDa, and the K_d is 2 mm. The residuals from the fit are shown in the lower panel. D, SARS-CoV N protein was cross-linked with various concentrations of BS³ (0–2 mm). The cross-linked products were analyzed by SDS-PAGE in an 8% gel. The arrows point to bands corresponding to monomer (M), dimer (D), tetramer (T), and higher order structures (H).

Fig. 4

Mapping the regions involved in dimerization.A, schematic diagram of different N protein constructs. B, cross-linking of N protein fragments NF1–4. Purified untagged proteins were treated with BS³ at 0, 1, 2, and 4 mm concentration. The reaction mixtures were fractionated by SDS-PAGE (10% for NF2, 12% for NF1, NF3, and NF4) and stained with Coomassie Blue. Bands corresponding to monomer and dimer are labeled as M and D, respectively.

Fig. 5

Inhibition of the full-length N protein dimerization.A, gel filtration profile (Superdex 200 16/30) of the mixture of full-length N and NF4 at 1:10 molar ratio (solid line). Peak A has an elution volume of 12.35 ml, and peak B elutes at 13.61 ml. The profile of the full-length N protein alone on the same column reveals a single elution peak at 11.32 ml (dotted line). AU, absorbance units. B, protein composition and cross-linking of the gel filtration elution fractions of N/NF4 mixture, visualized by silver staining after SDS-PAGE. Fractions of peaks A and B in panel A were treated with 1 mm BS³. Samples containing only full-length N protein or NF4 were also cross-linked and loaded on the same gel. N/N, full-length N protein homodimer; N/NF4, heterodimer of the full-length N and NF4; NF4/NF4, homodimer of NF4.