A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro

Abstract

Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2'-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0+/-0.4 nm, which consist of several (n=6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.

Fig. 1

AFM (a and c) and EM (b) images of ORF3 protein–fibrillarin complexes formed in vitro. (a and b) Mixtures of fibrillarin with wild-type ORF3 protein. (c) Mixture of fibrillarin with the ORF3 L149A mutant, which does not interact with fibrillarin [similar images were obtained when wild-type ORF3 protein was mixed with fibrillarin mutant lacking the GAR domain (Fib2ΔGAR), which does not interact with the ORF3 protein]. The recombinant ORF3 protein (ORF3–His; 10 ng) and fibrillarin were mixed in 15 μl of buffer A (10 mM Hepes–KOH, pH 7.6, 100 mM KCl) in a 1:1 molar ratio (15 ng/μl) and incubated at room temperature for 30 min. (a and c) For AFM, mixtures of fibrillarin with ORF3 protein were diluted to ∼ 5 ng/μl in deionized water and 5–10 μl was placed onto freshly cleaved mica strips for 5–15 min. The strips were rinsed with water and dried at room temperature. Imaging of complexes was done in tapping mode by using a Nanowizard® BioAFM (JPK, Berlin, Germany). Silicon beam cantilevers (Veeco Instruments Ltd., Cambridge, UK) with a nominal spring constant of 40 N/m and a resonant frequency of 300 kHz were used. All the AFM experiments were performed in air at room temperature, and the images were captured in constant height mode by using a scan speed of 0.5 Hz. Images, including two-dimensional (2d) and three-dimensional (3d) representations, were processed using JPK software and transferred to Adobe Photoshop for layout. Sample heights and lengths were measured automatically using the JPK software. Cross sections were made around the ring structures along lines connecting centers of granules in the anticlockwise direction starting from granule 1 (as indicated by arrows) to illustrate the heights of the complexes. Periodical height variations represent complexes containing six to eight granules arranged into ringlike structures. (b) For EM, complexes of the ORF3 protein and fibrillarin were negatively stained with 2% sodium phosphotungstate (pH 7.0). The specimens were examined and photographed in a Phillips CM 10 transmission electron microscope. Scale bars represent 20 nm.

Fig. 2

Analysis of the oligomeric state of the ORF3 protein–fibrillarin complexes. (a) Sedimentation distribution of the complexes. Samples of the ORF3–His and fibrillarin–His complexes were loaded onto the top of 10–30% sucrose gradients and centrifuged for 20 h in a Beckman SW41 rotor at 150,000g at 4 °C. Gradients were monitored for absorbance at 254 nm (A₂₅₄) during collection of the fractions from the top (fraction 1). The positions in the gradient of molecular mass markers (M), 45 kDa (albumin from chicken egg white), 200 kDa (β-amylase), 340 kDa (fibrinogen from bovine plasma), 545 kDa (urease from Jack bean), are shown. The presence of ringlike complexes in fractions 8–10 was confirmed by EM. (b) Distribution of the ORF3 protein and fibrillarin over gradient fractions determined by SDS-PAGE. The gels were stained with Coomassie brilliant blue, and protein bands were analyzed by densitometry using ImageJ software showing the equimolar ORF3 protein–fibrillarin ratio in the complexes (gradient fractions 8–10). Positions of the ORF3–His (ORF3) and fibrillarin–His (Fib) are shown on the right, and those of molecular mass markers are on the left. (c) Schematic representation of ORF3 protein–fibrillarin complex containing eight granules. Each single granule consists of one molecule of the ORF3 protein (ORF3) and one molecule of fibrillarin (Fib). The rings are formed by ORF3 protein–ORF3 protein association. Shapes and dimensions of molecules do not reflect real proportions.