Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein

Abstract

Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed.

Fig. 1

Bioinformatic analysis of GBNV N protein. a Schematic representation of predicted nucleic-acid-binding regions (underlined) and sites of phosphorylation (coloured residues) of the N protein. b Output obtained for the GBNV N protein using the FoldIndex program. The unfolded region in the protein has been boxed

Fig. 2

Expression in E. coli and purification of the GBNV N protein and its truncated forms. a Schematic representation of the full-length and deletion mutants of the N protein. Black boxes represent the regions that are present in each protein. b SDS-PAGE analysis of full-length N protein and truncated N proteins purified by Ni-NTA affinity chromatography. Lane 1, molecular mass markers; lanes 2, 3, 4, 5 and 6, full-length, C∆15, N∆20, C∆37 and N∆40 N protein respectively. c Western blot analysis of purified proteins using A10D10 monoclonal antibody to GBNV N protein [14]. Lanes are as marked in panel b

Fig. 3

Biophysical characterization of GBNV N protein. a CD spectrum of N protein. The far-UV CD spectrum for the N protein (0.1 mg/ml) was recorded using a Jasco-815 spectropolarimeter. The molar ellipticity was calculated using a subunit mass of 32 kDa. b Fluorescence spectrum of N protein. The fluorescence spectrum for the N protein (0.1 mg/ml) was recorded using a Perkin Elmer LS 55 luminescence spectrometer after excitation at 280 nm. c Thermal melting profile of N protein. The molar ellipticity (y-axis) was monitored at 208 nm as a function of temperature (x-axis) and plotted as shown. d Elution profile of N protein on a Superdex S-200 analytical gel filteration column. Peak 1 represents the void fraction (›600 kDa). Peaks 2 and 3 correspond to 128-kDa (tetramer) and 32-kDa (monomer) proteins, respectively

Fig. 4

RNA binding properties of recombinant N protein. a Purified N protein was run on a 0.6 % agarose gel and stained with ethidium bromide and Coomassie blue. Lanes 1 and 2 show the N protein (50 μg) in duplicate. b Analysis of RNA species associated with N protein on a 2% native agarose gel. Lane 1: RNA from N protein; lane 2: RNA size markers

Fig. 5

In vitro phosphorylation of GBNV N protein. a Phosphorylation of the N protein (5 μg) was carried out by mixing γ³²P ATP (1 μCi) and the soluble fraction of tobacco plant sap in 25 mM HEPES buffer, pH 7.2, containing 2 mM MnCl₂, incubation for 30 min at 30°C, and analysis by SDS-PAGE. The left panel shows an autoradiogram, and the right panel shows the same gel stained with Coomassie blue. Lane 1, N protein with plant sap; lane 2, N protein alone without plant sap (negative control); lane 3, plant sap alone without N protein (negative control), lane 4: molecular mass markers; lanes 5 and 6, reaction in the presence of 2 mM EGTA and 2 mM EDTA, respectively. b Effect of metal ions on phosphorylation of the N protein. The phosphorylation reaction was carried out without any metal ions (lane 1) or with varying concentrations (0.5, 1 and 2 mM) of MnCl₂ (lanes 2, 3, and 4, respectively). Similarly, the reaction was carried out separately with 0.5, 1 and 2 mM MgCl₂ (lanes 5-7) and CaCl₂ (lanes 8-10). Lane 11, molecular mass markers. The left panel shows an autoradiogram, and the right panel shows the same gel stained with Coomassie blue. c The phosphorylation reaction was carried out with 1 μCi of γ ³²P GTP as the phosphoryl group donor instead of labeled ATP. Lane 1, plant sap alone; lane 2, N protein alone; lane 3, reaction carried out in the absence of any metal ions; lanes 4, 5 and 6, reaction carried out in the presence of 2 mM MnCl₂, 2 mM MgCl₂ and 2 mM CaCl₂, respectively; lane 7, molecular mass markers. d Phosphorylation of the N protein (5 μg) was carried out with 200 ng of purified recombinant tobacco CK2 instead of plant sap as described in the legend to Fig. 5 a. Lane 1, CK2 alone without N protein; lane 2, N protein alone without CK2; lane 3, N protein with CK2; lane 4, molecular mass markers; lane 5, BSA (Calbiochem) with CK2 (negative control); lane 6, histone H1 (Sigma) with CK2 (positive control); lane 7, histone H1 (Sigma) without CK2. e Phosphorylation reaction carried out with 100 ng of purified recombinant chickpea CDPK instead of plant sap. Lane 1, CDPK alone without N protein (negative control); lane 2, N protein alone without CDPK; lane 3, N protein with CDPK; lane 4, molecular mass markers; lane 5, histone H1 (Sigma) with CDPK (positive control); lane 6, BSA (Calbiochem) with CDPK (negative control)