Functional characterization and subcellular localization of the 16K cysteine-rich suppressor of gene silencing protein of tobacco rattle virus

The 16 kDa cysteine-rich protein (16K) of tobacco rattle virus (TRV) is known to partially suppress RNA silencing in Drosophila cells. In this study, we show that 16K suppresses RNA silencing in green fluorescent protein (GFP)-transgenic Nicotiana benthamiana plants using an Agrobacterium-mediated transient assay. 16K slightly reduced the accumulation of short interfering RNAs (siRNA) of GFP, suggesting that the protein may interfere with the initiation and/or maintenance of RNA silencing. Deletion of either the N- or C-terminal part of 16K indicated that the entire 16K open reading frame (ORF) is necessary for its silencing suppression function. Pentapeptide insertion scanning mutagenesis (PSM) revealed that only two short regions of 16K tolerated five extra amino acid insertions without considerable reduction in its silencing suppression function. The tolerant regions coincide with sequence variability between tobravirus cysteine-rich proteins, indicating a strong functional and/or structural conservation of TRV 16K. Confocal laser scanning microscopy of transiently expressed 16K fusions to red fluorescent protein (RFP) revealed a predominant cytoplasmic localization and, in addition, a nuclear localization. In contrast, fusions of RFP with the N-terminal region of 16K localized exclusively to the cytoplasm, whereas fusions between RFP and the C-terminal region of 16K displayed an exclusive nuclear localization. Further analysis of 16K-derived peptide fusions demonstrated that the 16K C-terminal region contained at least two functional bipartite nuclear localization signals which were independently capable of nuclear targeting.

Download Full-text

Functional Characterization of Nuclear Localization Signals in Yeast Sm Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.7943-7954.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 7943-7954 ◽

Cited By ~ 27

Author(s):

Rémy Bordonné

Keyword(s):

Amino Acid ◽

Nuclear Localization ◽

Fluorescent Protein ◽

Functional Characterization ◽

Basic Amino Acid ◽

Core Complex ◽

Sm Proteins ◽

Nuclear Localization Signals ◽

Core Proteins ◽

Carboxyl Terminal

ABSTRACT In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.

Download Full-text

Wheat streak mosaic virus P1 Binds to dsRNAs without Size and Sequence Specificity and a GW Motif Is Crucial for Suppression of RNA Silencing

Viruses ◽

10.3390/v11050472 ◽

2019 ◽

Vol 11 (5) ◽

pp. 472 ◽

Cited By ~ 4

Author(s):

Adarsh K. Gupta ◽

Satyanarayana Tatineni

Keyword(s):

Mosaic Virus ◽

Rna Silencing ◽

Fluorescent Protein ◽

Wheat Streak Mosaic Virus ◽

Terminal Region ◽

Single Amino Acid ◽

Sequence Specificity ◽

Independent Manner ◽

Silencing Suppression

Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae) is an economically important virus infecting wheat in the Great Plains region of the USA. Previously, we reported that the P1 protein of WSMV acts as a viral suppressor of RNA silencing. In this study, we delineated the minimal region of WSMV P1 and examined its mechanisms in suppression of RNA silencing. We found that the 25 N-terminal amino acids are dispensable, while deletion of a single amino acid at the C-terminal region completely abolished the RNA silencing suppression activity of P1. Electrophoretic mobility shift assays with in vitro expressed P1 revealed that the P1 protein formed complexes with green fluorescent protein-derived 180-nt dsRNA and 21 and 24-nt ds-siRNAs, and WSMV coat protein-specific 600-nt dsRNA. These data suggest that the P1 protein of WSMV binds to dsRNAs in a size- and sequence-independent manner. Additionally, in vitro dicing assay with human Dicer revealed that the P1 protein efficiently protects dsRNAs from processing by Dicer into siRNAs, by forming complexes with dsRNA. Sequence comparison of P1-like proteins from select potyvirid species revealed that WSMV P1 harbors a glycine-tryptophan (GW) motif at the C-terminal region. Disruption of GW motif in WSMV P1 through W303A mutation resulted in loss of silencing suppression function and pathogenicity enhancement, and abolished WSMV viability. These data suggest that the mechanisms of suppression of RNA silencing of P1 proteins of potyvirid species appear to be broadly conserved in the family Potyviridae.

Download Full-text

RNA Silencing Suppression by a Second Copy of the P1 Serine Protease of Cucumber Vein Yellowing Ipomovirus, a Member of the Family Potyviridae That Lacks the Cysteine Protease HCPro

Journal of Virology ◽

10.1128/jvi.00985-06 ◽

2006 ◽

Vol 80 (20) ◽

pp. 10055-10063 ◽

Cited By ~ 80

Author(s):

Adrian Valli ◽

Ana Montserrat Martín-Hernández ◽

Juan José López-Moya ◽

Juan Antonio García

Keyword(s):

Rna Silencing ◽

Fluorescent Protein ◽

Serine Proteinase ◽

Plum Pox Virus ◽

Coding Region ◽

Long Distance ◽

Systemic Spread ◽

The Family ◽

Silencing Suppression ◽

Green Fluorescent

ABSTRACT The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.

Download Full-text

Nuclear localization of the Epstein–Barr virus EBNA3B protein

Journal of General Virology ◽

10.1099/vir.0.81640-0 ◽

2006 ◽

Vol 87 (4) ◽

pp. 789-793 ◽

Cited By ~ 7

Author(s):

Anita Burgess ◽

Marion Buck ◽

Kenia Krauer ◽

Tom Sculley

Keyword(s):

Nuclear Localization ◽

Epstein Barr Virus ◽

Fluorescent Protein ◽

Site Directed Mutagenesis ◽

Nuclear Antigen ◽

Nuclear Localization Signals ◽

Barr Virus ◽

Computer Analyses ◽

Epstein Barr ◽

Green Fluorescent

The Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

Download Full-text

Nuclear targeting of the β isoform of Type II phosphatidylinositol phosphate kinase (phosphatidylinositol 5-phosphate 4-kinase) by its α-helix 7

Biochemical Journal ◽

10.1042/bj3460587 ◽

2000 ◽

Vol 346 (3) ◽

pp. 587-591 ◽

Cited By ~ 50

Author(s):

Antonio CIRUELA ◽

Katherine A. HINCHLIFFE ◽

Nullin DIVECHA ◽

Robin F. IRVINE

Keyword(s):

Nuclear Localization ◽

Fluorescent Protein ◽

Physiological Role ◽

Nuclear Localization Sequence ◽

Type Ii ◽

Nuclear Targeting ◽

Phosphatidylinositol Phosphate ◽

Localization Sequence ◽

Α Helix ◽

Green Fluorescent

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIβ isoform. Type IIβ PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the α (cytosolic) and β PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of α-helix (α-helix 7) that is specific to the β isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIβ PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.

Download Full-text

A Newly Identified Virus in the Family Potyviridae Encodes Two Leader Cysteine Proteases in Tandem That Evolved Contrasting RNA Silencing Suppression Functions

Journal of Virology ◽

10.1128/jvi.01414-20 ◽

2020 ◽

Vol 95 (1) ◽

Author(s):

Li Qin ◽

Wentao Shen ◽

Zhongfa Tang ◽

Weiyao Hu ◽

Lingna Shangguan ◽

...

Keyword(s):

Rna Silencing ◽

Rna Viruses ◽

Evolutionary Relationship ◽

Cysteine Proteases ◽

Terminal Region ◽

Crop Damage ◽

Virus Species ◽

Protease Domain ◽

The Family ◽

Silencing Suppression

ABSTRACT Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability. IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

Download Full-text

Epstein–Barr virus nuclear antigen 3A contains six nuclear-localization signals

Journal of General Virology ◽

10.1099/vir.0.81927-0 ◽

2006 ◽

Vol 87 (10) ◽

pp. 2879-2884 ◽

Cited By ~ 7

Author(s):

Marion Buck ◽

Anita Burgess ◽

Roslynn Stirzaker ◽

Kenia Krauer ◽

Tom Sculley

Keyword(s):

Nuclear Localization ◽

Epstein Barr Virus ◽

Fluorescent Protein ◽

Viral Proteins ◽

Site Directed Mutagenesis ◽

Nuclear Antigen ◽

Nuclear Localization Signals ◽

Barr Virus ◽

Epstein Barr

The Epstein–Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein–Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix. By using enhanced green fluorescent protein-tagged deletion mutants of EBNA3A in combination with site-directed mutagenesis, an additional five functional nuclear-localization signals have been identified in the EBNA3A protein. Two of these (aa 63–66 and 375–381) were computer-predicted, whilst the remaining three (aa 394–398, 573–578 and 598–603) were defined functionally in this study.

Download Full-text

Dependence on Nuclear Localization Signals of the Opioid Growth Factor Receptor in the Regulation of Cell Proliferation

Experimental Biology and Medicine ◽

10.3181/0901-rm-16 ◽

2009 ◽

Vol 234 (5) ◽

pp. 532-541 ◽

Cited By ~ 27

Author(s):

Fan Cheng ◽

Patricia J. McLaughlin ◽

Michael F. Verderame ◽

Ian S. Zagon

Keyword(s):

Cell Proliferation ◽

Growth Factor ◽

Nuclear Localization ◽

Fluorescent Protein ◽

Growth Factor Receptor ◽

Full Length ◽

Nuclear Pore ◽

Nucleocytoplasmic Trafficking ◽

Nuclear Localization Signals ◽

Opioid Growth Factor

The opioid growth factor receptor (OGFr) mediates the inhibitory action of OGF on cell replication of normal and neoplastic cells. The spatiotemporal course of OGFr nucleocytoplasmic trafficking was determined with a probe of full-length OGFr fused to enhanced green fluorescent protein (eGFP). Translation of OGFr required 8.5 hours, and transit into the nucleus required 8 hours; OGFr remained in the nucleus for 8 days. OGFr was initially expressed on the outer nuclear envelope, transited to the paranuclear cytoplasm, and into the nucleus. Transport through the nuclear pore was elucidated by mutation of the nuclear localization signal (NLS) sequences in full-length OGFr. Mutation of each NLS reduced nuclear localization by 5%–50%, whereas simultaneous mutation of NLS383–386 and NLS456–460 abolished OGFr-eGFP nuclear localization in 80% of the cells. To determine whether intact NLSs are important for the inhibition of cell proliferation, DNA synthesis was monitored with BrdU. Wild-type OGFr-eGFP–transfected cells had 20% BrdU-positive cells, whereas cells with simultaneous mutation of all three NLS sites had a 70% labeling index. These results indicate that the regulation of cell proliferation by the OGF-OGFr axis is dependent on nucleocytoplasmic translocation and reliant on the integrity of two NLSs in OGFr to interact with transport receptors.

Download Full-text

The Benyvirus RNA Silencing Suppressor Is Essential for Long-Distance Movement, Requires Both Zinc-Finger and NoLS Basic Residues but Not a Nucleolar Localization for Its Silencing-Suppression Activity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-12-0142-r ◽

2013 ◽

Vol 26 (2) ◽

pp. 168-181 ◽

Cited By ~ 32

Author(s):

Sotaro Chiba ◽

Kamal Hleibieh ◽

Alice Delbianco ◽

Elodie Klein ◽

Claudio Ratti ◽

...

Keyword(s):

Zinc Finger ◽

Rna Silencing ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Nucleolar Localization ◽

Silencing Suppressor ◽

Long Distance ◽

Microscopy Imaging ◽

Silencing Suppression ◽

Long Distance Movement

The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.

Download Full-text

Virus-induced gene silencing in the perennial woody Paeonia ostii

PeerJ ◽

10.7717/peerj.7001 ◽

2019 ◽

Vol 7 ◽

pp. e7001

Author(s):

Lihang Xie ◽

Qingyu Zhang ◽

Daoyang Sun ◽

Weizong Yang ◽

Jiayuan Hu ◽

...

Keyword(s):

Gene Silencing ◽

Fluorescent Protein ◽

Molecular Genetic ◽

Functional Characterization ◽

Tobacco Rattle Virus ◽

Vacuum Infiltration ◽

Genomic Research ◽

Virus Induced Gene Silencing ◽

Tree Peony ◽

Medicinal Value

Tree peony is a perennial deciduous shrub with great ornamental and medicinal value. A limitation of its current functional genomic research is the lack of effective molecular genetic tools. Here, the first application of a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) in the tree peony species Paeonia ostii is presented. Two different approaches, leaf syringe-infiltration and seedling vacuum-infiltration, were utilized for Agrobacterium-mediated inoculation. The vacuum-infiltration was shown to result in a more complete Agrobacterium penetration than syringe-infiltration, and thereby determined as an appropriate inoculation method. The silencing of reporter gene PoPDS encoding phytoene desaturase was achieved in TRV-PoPDS-infected triennial tree peony plantlets, with a typical photobleaching phenotype shown in uppermost newly-sprouted leaves. The endogenous PoPDS transcripts were remarkably down-regulated in VIGS photobleached leaves. Moreover, the green fluorescent protein (GFP) fluorescence was detected in leaves and roots of plants inoculated with TRV-GFP, suggesting the capability of TRV to silence genes in various tissues. Taken together, the data demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in tree peony.

Download Full-text