Genetic dissection of a putative nucleolar localization signal in the coat protein of ourmia melon virus

Rossi, Marika; Genre, Andrea; Turina, Massimo

doi:10.1007/s00705-013-1923-0

Cited by 10 publications

(9 citation statements)

References 27 publications

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“…Well-characterized plant virus proteins of this category include the ORF3 protein of Groundnut rosette virus (GRV; unassigned family, genus Umbravirus), the CP of Potato leaf roll virus (family Luteoviridae), the genome-linked viral protein of Potato virus A (family Potyviridae), the 2b suppressor of RNA silencing of Cucumber mosaic virus (family Bromoviridae), the P7a movement protein of Beet black scorch virus (family Tombusviridae), the CP of Alfalfa mosaic virus (family Bromoviridae), the P30 movement protein encoded by Turnip vein clearing virus (family Virgaviridae), and the P14 suppressor of RNA silencing and P25 of Beet necrotic yellow vein virus (family Virgaviridae; Haupt et al, 2005b;Kim et al, 2007;Rajamäki and Valkonen, 2009;González et al, 2010;Herranz et al, 2012;Wang et al, 2012;Chiba et al, 2013;Levy et al, 2013). Other viral proteins found in the nucleolus include the P3 protein of Tobacco etch virus (family Potyviridae) and the CP of Ourmia melon virus (unassigned family, genus Ourmiavirus), but the function of these nucleolar localizations is unknown (Taliansky et al, 2010;Crivelli et al, 2011;Rossi et al, 2014). Another nucleolus-localized …”

Section: Discussionmentioning

confidence: 99%

Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement, Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana

et al. 2015

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Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-a family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-a paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-a in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-a-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-a interaction in plants, nucleolar targeting, and long-distance movement.

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Section: Discussionmentioning

confidence: 99%

Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement, Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana

et al. 2015

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“…A link between the ability to interact with the nucleolus and the outcome of viral infection has been established in many cases (Hiscox, 2007;Salvetti and Greco, 2014). In recent years, diverse plant viruses belonging to different genera or families have also been found to encode one or multiple proteins localizing to the nucleolus (Martínez and Daròs, 2014;Rossi et al, 2014;Taliansky et al, 2010;and references therein). In the nucleolus, Groundnut rosette virus (GRV, an umbravirus) open reading frame 3 (ORF3) interacts with fibrillarin and recruits this nucleolar protein to form infectious ribonucleoproteins (RNPs) in the cytoplasm.…”

Section: Introductionmentioning

confidence: 99%

“…For most other plant viruses, the rationale for their nucleolus targeting is currently unclear. However, a growing number of correlative studies have indicated that the nucleolus may play a role in many steps in the infection cycle of these viruses (Martínez and Daròs, 2014;Rossi et al, 2014;Taliansky et al, 2010;and references therein).…”

Section: Introductionmentioning

confidence: 99%

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

Zheng

Chang-hu

et al. 2015

Molecular Plant Pathology

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Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2-YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2-YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2-YFP fluorescence were affected in fibrillarin-silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.

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“…Such intracellular targeting seems to be determined by a short stretch of amino acids at the N-terminal region of the protein. This region is rich in lysine and arginine residues and mutations of only three amino acids within this region resulted in the complete exclusion of the GFP-CP mutant from the nucleolus (Rossi et al 2014). These results provide a model whereby the OuMV CP has a functional activity in addition to its structural role.…”

mentioning

confidence: 74%

“…benthamiana cells (Rossi et al 2014). In the present work, we used some of the most significant CP mutants from that study (Fig.…”

mentioning

confidence: 99%

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense

Rossi¹,

Vallino²,

Abbà³

et al. 2015

MPMI

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The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV 6710 , with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV 6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CP wt , in situ immunolocalization experiments indicated that CP 6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.Ourmia melon virus (OuMV) is the best-characterized species of the Ourmiaviridae family, which also consists of Epirus cherry virus (EpCV) and Cassava virus C (CaVC) species (King et al. 2012). Three plus-strand RNA molecules form the OuMV genome and each segment encodes a single protein: RNA1 directs synthesis of an RNA-dependent RNA polymerase (RdRp), RNA2 encodes a movement protein (MP), and RNA3 specifies the 22-kDa coat protein (CP) (Rastgou et al. 2009). Ourmiaviruses have affinities with other postive single stranded RNA multipartite viruses, such as the members of the family Bromoviridae, but their unique structural and molecular features have resulted in their classification in a novel viral family. In fact, phylogenetic analyses showed that the members of the family likely went through a very unusual evolutionary process based on a possible reassortment between a plant virus and a mycovirus. In particular, OuMV RdRp shares distant similarity only with RdRp of narnaviruses, a group of viruses which mainly infects fungi; however, the MP is distantly related to MP of plant tombusviruses. The CP has limited but significant similarity with CP of plant and mammalian viruses, although its phylogenetic origin remains uncertain (Rastgou et al. 2009). Another peculiarity of the OuMV CP is the unique morphology of the virions among plant viruses: bacilliform particles of three different lengths with pointed ends (Lisa et al. 1988). Ectopic expression of RNA3 in Nicotiana benthamiana does not result in virion formation because the active replication of OuMV is necessary fo...

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Genetic dissection of a putative nucleolar localization signal in the coat protein of ourmia melon virus

Cited by 10 publications

References 27 publications

Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement, Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana

Importin-α-Mediated Nucleolar Localization of Potato Mop-Top Virus TRIPLE GENE BLOCK1 (TGB1) Protein Facilitates Virus Systemic Movement, Whereas TGB1 Self-Interaction Is Required for Cell-to-Cell Movement in Nicotiana benthamiana

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense

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