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. 2007 Jun 26;104(26):11115-20.
doi: 10.1073/pnas.0704632104. Epub 2007 Jun 18.

Interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection

Sang Hyon Kim  1 , Stuart Macfarlane, Natalia O Kalinina, Daria V Rakitina, Eugene V Ryabov, Trudi Gillespie, Sophie Haupt, John W S Brown, Michael Taliansky
Affiliations

Interaction of a plant virus-encoded protein with the major nucleolar protein fibrillarin is required for systemic virus infection

Sang Hyon Kim et al. Proc Natl Acad Sci U S A. .

Abstract

The nucleolus and specific nucleolar proteins are involved in the life cycles of some plant and animal viruses, but the functions of these proteins and of nucleolar trafficking in virus infections are largely unknown. The ORF3 protein of the plant virus, groundnut rosette virus (an umbravirus), has been shown to cycle through the nucleus, passing through Cajal bodies to the nucleolus and then exiting back into the cytoplasm. This journey is absolutely required for the formation of viral ribonucleoprotein particles (RNPs) that, themselves, are essential for the spread of the virus to noninoculated leaves of the shoot tip. Here, we show that these processes rely on the interaction of the ORF3 protein with fibrillarin, a major nucleolar protein. Silencing of the fibrillarin gene prevents long-distance movement of groundnut rosette virus but does not affect viral replication or cell-to-cell movement. Repressing fibrillarin production also localizes the ORF3 protein to multiple Cajal body-like aggregates that fail to fuse with the nucleolus. Umbraviral ORF3 protein and fibrillarin interact in vitro and, when mixed with umbravirus RNA, form an RNP complex. This complex has a filamentous structure with some regular helical features, resembling the RNP complex formed in vivo during umbravirus infection. The filaments formed in vitro are infectious when inoculated to plants, and their infectivity is resistant to RNase. These results demonstrate previously undescribed functions for fibrillarin as an essential component of translocatable viral RNPs and may have implications for other plant and animal viruses that interact with the nucleolus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Correlation between the ability of the ORF3 protein to traffic through the nucleolus and the relocalization of fibrillarin, formation of viral RNPs, and long-distance movement. Wild-type and mutant ORF3 protein sequences of the R-rich and L–rich domains are shown in combination with data on nuclear (N) and nucleolar (No) localization, nuclear export (N-exp) of the ORF3 protein, relocalization of fibrillarin (Cyt. fibrillarin), RNP formation, and virus long-distance movement (LDM) (14).
Fig. 2.
Fig. 2.
Virus-induced silencing of the fibrillarin gene (NbFib) in N. benthamiana plants by using a TRV vector. (A–C) Expression of fibrillarin was suppressed by TRV-NbFib to different levels, and plants with effectively three different phenotypes (I, II, and III) were generated. (A) Growth phenotypes of silenced plants in comparison with control (c) nonsilenced plants. (B) Semiquantitative RT-PCR analysis of NbFib mRNA accumulation. Ethidium bromide-stained agarose gels show RT-PCR products corresponding to the fragments of NbFib (Fib) mRNA (320 bp) and ubiquitin mRNA (176 bp) used as a control, as indicated by arrows. Lanes 1–3 represent plant replicates. (C) Western blot analysis of fibrillarin (Fib) accumulation. The position of fibrillarin is shown by an arrow. Lanes 1–3 as above. (D) Immunofluorescence staining of cells of fibrillarin-silenced (group II; Fib-s) and nonsilenced (Non-s) plants with fibrillarin and coilin antibodies visualized by confocal microscopy. Fibrillarin levels are significantly reduced in the nucleolus and are undetectable in CBs, visualized by using antibody against coilin. (Scale bars, 5 μm.)
Fig. 3.
Fig. 3.
Fibrillarin knockdown suppresses long-distance movement of GRV and nucleolar localization of ORF3 protein. (A and B) Accumulation of GRV-YB in inoculated (in) and uninoculated (u) leaves of nonsilenced (Non-s) and fibrillarin-silenced (Fib-s) N. benthamiana plants of group II (A, B, and Table 2). (A) Symptoms on systemically infected, uninoculated leaves. Nonsilenced plants show yellow blotch symptoms characteristic of the GRV-YB isolate, whereas fibrillarin-silenced plants (group II) are symptomless. (B) Northern blot analysis of RNA isolated from leaves of the infected plants. GRV RNA is shown by an arrow; rRNA bands were stained by ethidium bromide for loading control (lower blot). (C) Localization of the ORF3-GFP protein delivered by Agrobacterium into cells of the nonsilenced and silenced plants. No, nucleolus; CBL, Cajal body-like structures; N, nuclei (shown by dashed lines according to DAPI staining). (Scale bars, 5 μm.)
Fig. 4.
Fig. 4.
Fibrillarin interacts with ORF3 protein. (A) Representation of Arabidopsis fibrillarin 2 (Fib2) showing GAR, RNA binding and α-helix domains, the GST fusion protein (GST-Fib2), GST fused with Fib2 fragment (GST-Fib2*) and the GAR deletion mutant (GST-Fib2ΔGAR). Numbers are amino acid residue positions; triangle shows position of thrombin cleavage site. (B) Far Western blot analysis of the interaction between ORF3 protein and fibrillarin, Fib2. Blots containing GST-Fib2 (also containing GST-Fib2*; lane 1), GST-Fib2ΔGAR (lane 2), GST (lane 3) and Fib2 (GST-Fib2 treated with thrombin; lane 4) were incubated with or without the ORF3 protein [+ORF3 (Left) and −ORF3 (Center)] and probed by anti-ORF3 antibody. (Right) Coomassie staining. Positions of GST-Fib2, GST-Fib2*, and Fib2 are shown on the left and those of molecular mass markers are on the right. (C) Far Western blot analysis of the interaction between the purified recombinant ORF3 protein mutants and fibrillarin. Blots containing GST-Fib2 (and GST-Fib2*) were incubated with wild-type (1) or mutant ORF3 proteins (LA, 2; L149A, 3; L153A, 4; RA, 5) and probed as above. Lane 7 represents Ponceau S staining of fibrillarin. Lane 6 corresponds to a control sample prepared from a plant infected with the TMV vector alone by using the same protocol as for the recombinant ORF3 proteins. The lower blot shows expression levels of wild-type and mutant ORF3 proteins (Coomassie staining) as indicated. M, marker of 30 kDa.
Fig. 5.
Fig. 5.
EM images showing complexes generated by different mixtures of umbraviral RNA, ORF3 protein, and fibrillarin. (A) Complex of GRV ORF3 protein and PEMV-2 RNA (F, thin filaments; DC, densely coated RNA). (B) Complex of Arabidopsis fibrillarin (Fib2) and PEMV-2 RNA (F and DC as above). (C) Disk-like complexes formed by ORF3 protein and Fib2 in the absence of RNA. (D) Complexes of ORF3 protein, Fib2 and RNA (an arrow shows elements of helical structure). Insets show higher magnification. (Scale bars: A–D, 100 nm; Insets, 50 nm.)
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