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

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 longdistance 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 bodylike 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.Cajal bodies ͉ plant virus movement ͉ ribonucleoprotein particles T he nucleolus is a subnuclear domain and is the site of transcription and processing of rRNA and of ribosome biogenesis. In addition, the nucleolus also participates in other aspects of RNA metabolism and cell function (1, 2). The nucleolus is structurally and functionally associated with Cajal bodies (CBs), which are structures found in both animals and plants (3, 4). CBs contain different proteins including coilin, a protein essential for CB formation, and fibrillarin, a major nucleolar protein that is a core component of small nucleolar ribonucleoprotein particles (snoRNPs) and is required for rRNA processing (4-7). CBs are involved in the maturation of small nuclear RNPs (snRNPs) and snoRNPs, which traffic through CBs before accumulating in splicing speckles and the nucleolus, respectively (8, 9). Both the nucleolus and CBs have a role in RNA silencing in plants (10,11). Finally, a number of animal and plant viruses including the RNAcontaining tobacco etch virus and the DNA-containing tomato yellow leaf curl virus have a nucleolar phase in their life cycle (12, 13). Recently, we have shown that the ability of the umbravirus, groundnut rosette virus (GRV), to move long distances through the phloem, the specialized vascular system used by plants for the transport of assimilates and macromolecules, depends strictly on the...

Control of Nuclear and Nucleolar Localization of Nuclear Inclusion Protein a of Picorna-LikePotato virus AinNicotianaSpecies

“…Although TEV NIa has been observed in the nucleolus (Schaad et al, 1996), detailed studies on nucleolar targeting of NIa have not been reported. Thus, our current findings on nucleolar and nuclear localization of a potyviral protein were made in a study area that has generally gained little attention in plant virology (Kim et al, 2007a(Kim et al, , 2007b.…”

“…The ORF3 protein of GRV (Kim et al, 2007a) and some proteins of animal viruses interact with fibrillarin or certain other nucleolar proteins (reviewed in Hiscox, 2007), which prompted us to test whether PVA NIa could interact with fibrillarin. Two copies (Nb Fib2a and Nb Fib2b) …”

“…To examine whether fibrillarin plays a role in PVA infection, as it does in infection of GRV (Kim et al, 2007a), fibrillarin expression was suppressed in N. benthamiana plants using a Tobacco rattle virus (TRV)-based vector carrying a fragment of the Nb Fib2a gene of N. benthamiana . N. benthamiana plants were also inoculated with an empty TRV vector as a control.…”

“…In plant cells infected with Groundnut rosette virus (GRV; genus Umbravirus), the GRV ORF3 protein enters the nucleus and accumulates in the nucleolus and Cajal bodies (CBs), small subnuclear bodies, or structures (Ryabov et al, 2004;Kim et al, 2007b). This viral protein interacts with the nucleolar protein fibrillarin, which is required for long-distance viral movement in plants (Kim et al, 2007a(Kim et al, , 2007bCanetta et al, 2008).…”

Nucleoli: Composition, Function, and Dynamics

“…In the best-studied system to date, the ORF3 long-distance movement protein of Groundnut rosette virus trafficks to the nucleolus via CBs, causing reorganization of CBs into multiple CBlike structures that fuse with the nucleolus. ORF3 then recruits fibrillarin (an abundant nucleolar RNA binding protein, known to be an RNA methylase) for assembly of cytoplasmic infectious viral particles (Kim et al, 2007a(Kim et al, , 2007bCanetta et al, 2008). The nucleolar localization of ORF3 is essential for systemic infection.…”