Cucumber mosaic virus 2b directs fibrillarin translocation to plasmodesmata to promote viral movement

Abstract

Cucumber mosaic virus (CMV) is one of the most widespread and infectious plant viruses affecting over 1200 plant species, including both monocots and dicots (Palukaitis et al., 1992; Mochizuki & Ohki, 2011). The CMV genome consists of three positive-stranded RNAs encoding five proteins: 1a, 2a, 2b, 3a, and coat protein (CP; Jacquemond, 2012). Among them, the multifunctional 2b protein regulates diverse processes throughout the viral life cycle, including viral movement both locally and systemically (Nemes et al., 2014), symptom development (Lewsey et al., 2009), and suppression of RNA silencing as part of the host defense response (Ji & Ding, 2001; Zhang et al., 2006; Zhou et al., 2014).

The 2b proteins of subgroup IA CMV strains, including Fenny Dale (Fny)-CMV strain and Shangdong (SD)-CMV strain, are known to partition between the nucleus and the cytoplasm, yet the biological relevance of such phenomena remains uncertain. Nuclear targeting of 2b proteins from subgroup IA strains is governed by two nuclear localization signals (NLSs), NLS1 and NLS2 (Wang et al., 2004; González et al., 2010; Duan et al., 2012). It was previously shown that NLS mutations impaired 2b's functions in RNA silencing suppression and virus pathogenicity (Lucy et al., 2000; Wang et al., 2004; Lewsey et al., 2009; González et al., 2010). Interestingly, other studies suggested that Fny2b fused with a nuclear export signal, which inhibits its sustained nuclear accumulation, still retains virus silencing suppressor (VSR) activity, suggesting that nuclear localization is not strictly required for this function (González et al., 2012). On the other hand, increasing 2b's nuclear accumulation can reduce its VSR activity while enhancing viral virulence (Du et al., 2014). These findings suggest that the NLS motifs may have broader implications in 2b functionality, potentially beyond its role in VSR activity.

Emerging evidence indicates that the ability of certain viral proteins to form liquid–liquid phase-separated (LLPS) condensates is essential for multiple aspects of the viral lifestyle, including enhancing replication, movement, and host manipulation (Etibor et al., 2021; May, 2024). These condensates act as dynamic compartments, organizing viral and host components to enhance the efficiency of viral gene functions. Liquid–liquid phase-separated-driven interactions between viral proteins and host factors have been shown to facilitate the systemic infection of various plant viruses (Brown et al., 2021). Nevertheless, whether CMV-2b proteins are capable of forming condensates and to what extent such capabilities contribute to its dynamic interactions with host factors and, moreover, to the viral pathogenicity remain largely unknown.

Fibrillarin (FIB), a key plant nucleolar protein involved in RNA processing, has been implicated in regulating the movements of different RNA viruses (Canetta et al., 2008; Hipper et al., 2013; Li et al., 2018; Decle-Carrasco et al., 2021). In Arabidopsis thaliana (hereafter Arabidopsis), silencing of FIB2 impeded the long-distance transport of umbravirus, groundnut rosette virus (GRV) (Souza & Carvalho, 2019). Together with GRV-ORF3 protein, FIB2 associates with viral RNA to form Ribonucleoprotein (RNP) particles, which are then transported from cell to cell, ultimately completing long-distance trafficking (Kim et al., 2007). Similarly, the P26 movement protein from Pea enation mosaic virus 2 (PEMV2) interacts with FIB2, forming droplets with PEMV2 genomic RNAs in vitro for systemic virus movement (Brown et al., 2021). These examples suggest that phase-separated droplets play an important role in virus–host interactions. However, the role of FIB2 protein during CMV infection, particularly in the context of viral protein interactions, remains to be determined.

Here, we show that CMV-2b forms condensates in planta, which are driven by its NLS motifs, and these two NLS domains are crucial for interaction of 2b with FIB2 proteins. Additionally, FIB2 proteins positively regulate CMV infection lifecycle and are required for 2b protein-dependent LLPS formation in vivo. Our findings reveal that the formation of 2b condensates and the translocation of 2b/FIB2 complex to the plasmodesmata (PD) are essential for facilitating viral cell-to-cell movement and promoting CMV infection.

Our data revealed that the CMV-2b protein directly interacts with plant fibrillarin and forms biomolecular condensates in nuclei in an NLS-dependent manner. Notably, these condensates do not contain coilin and therefore are distinct from the Cajal bodies. CMV-2b facilitates the nuclear export of FIB2 during CMV infection, and this nuclear-cytoplasmic re-localization of FIB2 is critical for the infection process. Genetic evidence from both Arabidopsis and Nicotiana further demonstrates that CMV leverages host FIB2 proteins to promote the efficient spread of the virus.

From a mechanism perspective, we show that FIB2 enhances the cell-to-cell movement of CMV-2b and facilitates its LLPS in vivo. The disruption of FIB2 function significantly reduces 2b condensate formation in nuclei and impairs its intercellular movement. These findings suggest that phase separation of 2b, supported by FIB2, is essential for the systemic spread of CMV. Furthermore, the reduced infectivity and mild symptoms associated with the CMV-2b∆NLS1/2 mutant strain, as previously reported (Lewsey et al., 2009), can now be attributed not only to the loss of its silencing suppressor activity but also to the impaired cell-to-cell movement caused by its disrupted condensate formation.

Collectively, our work identified plant FIB2 proteins as essential host factors that facilitate CMV infection and uncovered the molecular mechanism underlying the CMV-2b-FIB2 interaction. By demonstrating the role of phase separation in viral movement, this study provides new insights into the interplay between host nucleolar proteins and plant viruses, advancing our understanding of viral pathogenesis and host–virus interactions.

None declared.

DZ and NHC designed the experiments. DZ and HYX executed the experiments. DZ and NHC wrote the manuscript.

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