Coat proteins of Rice tungro bacilliform virus and Mungbean yellow mosaic virus contain multiple nuclear-localization signals and interact with importin α

Transport of the viral genome into the nucleus is an obligatory step in the replication cycle of plant pararetro- and geminiviruses. In both these virus types, the multifunctional coat protein (CP) is thought to be involved in this process. Here, a green fluorescent protein tagging approach was used to demonstrate nuclear import of the CPs of Rice tungro bacilliform virus (RTBV) and Mungbean yellow mosaic virus - Vigna (MYMV) in Nicotiana plumbaginifolia protoplasts. In both cases, at least two nuclear localization signals (NLSs) were identified and characterized. The NLSs of RTBV CP are located within both N- and C-terminal regions (residues 479KRPK/497KRK and 744KRK/758RRK), and those of MYMV CP within the N-terminal part (residues 3KR and 41KRRR). The MYMV and RTBV CP NLSs resemble classic mono- and bipartite NLSs, respectively. However, the N-terminal MYMV CP NLS and both RTBV CP NLSs show peculiarities in the number and position of basic residues. In vitro pull-down assays revealed interaction of RTBV and MYMV CPs with the nuclear import factor importin α, suggesting that both CPs are imported into the nucleus via an importin α-dependent pathway. The possibility that this pathway could serve for docking of virions to the nucleus is discussed.

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005.Efficiency of nuclear import of the chromatin-remodelling factor SRY is critical for sex determination

Reproduction Fertility and Development ◽

10.1071/srb05abs005 ◽

2005 ◽

Vol 17 (9) ◽

pp. 64

Author(s):

D. A. Jans ◽

G. Kaur ◽

I. K. H. Poon ◽

A. Delluc-Clavieries ◽

K. M. Wagstaff

Keyword(s):

Sex Determination ◽

Nuclear Localization ◽

Nuclear Import ◽

Sex Reversal ◽

Nuclear Accumulation ◽

Missense Mutations ◽

Nuclear Localization Signals ◽

Testicular Cells ◽

Xy Sex Reversal

15% of cases of human XY sex reversal are due to mutations in SRY (sex determining region on the Y chromosome), many of which map to one of SRY’s two independently acting nuclear localization signals (NLSs) flanking its DNA binding domain. The C-terminal NLS (C-NLS) targets SRY to the nucleus through a ‘conventional’ pathway dependent on the nuclear import receptor importin-β (Imp-β). No importin has been shown to bind the N-terminal NLS (N-NLS), but it is known to interact with the Ca2+-binding protein calmodulin (CaM). We examined seven distinct missense mutations in the SRY NLSs from XY sex-reversed human females for effects on nuclear import and ability to interact with CaM/Imp-β1. All mutations were found to result in reduced nuclear localization in transfected testicular cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce SRY nuclear accumulation, indicating a dependence of SRY nuclear import on CaM. Intriguingly, N-NLS mutants were resistant to CDZ’s effects, implying a loss of interaction with CaM; this was confirmed directly by in vitro binding experiments using recombinantly expressed protein. Either impaired CaM or Imp-β1 binding can thus be the basis of sex-reversal in human patients. Our results implicate a CaM-dependent nuclear import pathway for SRY mediated by the N-NLS that, together with the C-NLS, is required to achieve threshold levels of SRY in the nucleus for male sex determination.

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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.

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The Arginine-Rich Domains Present in Human Immunodeficiency Virus Type 1 Tat and Rev Function as Direct Importin β-Dependent Nuclear Localization Signals

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1210 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1210-1217 ◽

Cited By ~ 274

Author(s):

Ray Truant ◽

Bryan R. Cullen

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Localization Signals ◽

Immunodeficiency Virus ◽

Importin Α

ABSTRACT Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as targets for the importin α (Imp α) NLS receptor. Imp α is in turn bound by importin β (Imp β), which targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp α and instead bind directly to Imp β. Using in vitro nuclear import assays, we demonstrate that Imp α is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp β proved both sufficient and necessary, in that other β-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the target sites on Imp β for Imp α, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp β is also similar to Imp α binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct targets for Imp β and that therefore function independently of Imp α.

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SignalsThat Dictate Nuclear Localization of Human Papillomavirus Type 16Oncoprotein E6 in LivingCells

Journal of Virology ◽

10.1128/jvi.77.24.13232-13247.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13232-13247 ◽

Cited By ~ 36

Author(s):

Mingfang Tao ◽

Michael Kruhlak ◽

Shuhua Xia ◽

Elliot Androphy ◽

Zhi-Ming Zheng

Keyword(s):

Human Papillomavirus ◽

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Protein ◽

Fluorescent Protein ◽

Present Report ◽

Cell Cycle Control ◽

Point Mutations ◽

Coding Region ◽

Nuclear Localization Signals

ABSTRACT Human papillomavirus (HPV) type 16 E6 (16E6) is an oncogenic, multifunctional nuclear protein that induces p53 degradation and perturbs normal cell cycle control, leading to immortalization and transformation of infected keratinocytes and epithelial cells. Although it is unclear how 16E6 disrupts the epigenetic profile of host genes, its presence in the nucleus is a key feature. The present report describes intrinsic properties of 16E6 that influence its nuclear import in living cells. When the coding region of full-length 16E6 was inserted in frame into the C terminus of green fluorescent protein (GFP), it effectively prevented the 16E6 pre-mRNA from being spliced and led to the expression of a GFP-E6 fusion which localized predominantly to the nucleus. Further studies identified three novel nuclear localization signals (NLSs) in 16E6 that drive the protein to accumulate in the nucleus. We found that all three NLS sequences are rich in positively charged basic residues and that point mutations in these key residues could abolish the retention of 16E6 in the nucleus as well as the p53 degradation and cell immortalization activities of the protein. When inserted into corresponding regions of low-risk HPV type 6 E6, the three NLS sequences described for 16E6 functioned actively in converting the normally cytoplasmic HPV type 6 E6 into a nuclear protein. The separate NLS sequences, however, appear to play different roles in nuclear import and retention of HPV E6. The discovery of three unique NLS sequences in 16E6 provides new insights into the nuclear association of 16E6 which may reveal other novel activities of this important oncogenic protein.

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Functional analysis of the Cucumber mosaic virus 2b protein: pathogenicity and nuclear localization

Journal of General Virology ◽

10.1099/vir.0.80250-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 3135-3147 ◽

Cited By ~ 54

Author(s):

Yongzeng Wang ◽

Tzvi Tzfira ◽

Victor Gaba ◽

Vitaly Citovsky ◽

Peter Palukaitis ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Nuclear Localization ◽

Zucchini Yellow Mosaic Virus ◽

Yellow Mosaic Virus ◽

Silencing Suppressor ◽

Nuclear Localization Signals ◽

Systemic Movement ◽

2B Protein ◽

Function Expression

The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown to be a silencing suppressor and pathogenicity determinant in solanaceous hosts, but a movement determinant in cucumber. In addition, synergistic interactions between CMV and Zucchini yellow mosaic virus (ZYMV) have been described in several cucurbit species. Here, it was shown that deletion of the 2b gene from CMV prevented extensive systemic movement of the virus in zucchini squash, which could not be complemented by co-infection with ZYMV. Thus, ZYMV expressing a silencing suppressor with a different target could not complement the CMV 2b-specific movement function. Expression of the 2b protein from an attenuated ZYMV vector resulted in a synergistic response, largely restoring infection symptoms of wild-type ZYMV in several cucurbit species. Deletion or alteration of either of two nuclear localization signals (NLSs) did not affect nuclear localization in two assays, but did affect pathogenicity in several cucurbit species, whilst deletion of both NLSs led to loss of nuclear localization. The 2b protein interacted with an Arabidopsis thaliana karyopherin α protein (AtKAPα) in the yeast two-hybrid system, as did each of the two single NLS-deletion mutants. However, 2b protein containing a deletion of both NLSs was unable to interact with AtKAPα. These data suggest that the 2b protein localizes to the nucleus by using the karyopherin α-mediated system, but demonstrate that nuclear localization was insufficient for enhancement of the 2b-mediated pathogenic response in cucurbit hosts. Thus, the sequences corresponding to the two NLSs must have another role leading to pathogenicity enhancement.

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Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish

Biochemistry and Cell Biology ◽

10.1139/o97-044 ◽

1997 ◽

Vol 75 (5) ◽

pp. 633-640 ◽

Cited By ~ 24

Author(s):

Philippe Collas ◽

Peter Aleström

Keyword(s):

Nuclear Localization ◽

Plasmid Dna ◽

Nuclear Import ◽

Protein Import ◽

Nuclear Transport ◽

Nuclear Targeting ◽

Nuclear Localization Signals ◽

Expression Frequency ◽

Cytoplasmic Injection

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

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Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions

Journal of General Virology ◽

10.1099/vir.0.82021-0 ◽

2006 ◽

Vol 87 (9) ◽

pp. 2709-2720 ◽

Cited By ~ 7

Author(s):

Gideon Kass ◽

Gabriel Arad ◽

Joseph Rosenbluh ◽

Yedidya Gafni ◽

Adolf Graessmann ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Synthetic Peptides ◽

Mammalian Cells ◽

Specific Binding ◽

Nuclear Localization Signals ◽

Direct Demonstration ◽

Bipartite Genome ◽

Monopartite Genome

The plant-infecting geminiviruses deliver their genome and viral proteins into the host cell nucleus. Members of the family Geminiviridae possess either a bipartite genome composed of two ∼2.6 kb DNAs or a monopartite genome of ∼3.0 kb DNA. The bipartite genome of Bean dwarf mosaic virus (BDMV) encodes several karyophilic proteins, among them the capsid protein (CP) and BV1 (nuclear shuttle protein). A CP is also encoded by the monopartite genome of Tomato yellow leaf curl virus (TYLCV). Here, an in vitro assay system was used for direct demonstration of nuclear import of BDMV BV1 and TYLCV CP, as well as synthetic peptides containing their putative nuclear localization signals (NLSs). Full-length recombinant BDMV BV1 and TYLCV CP mediated import of conjugated fluorescently labelled BSA molecules into nuclei of permeabilized mammalian cells. Fluorescently labelled and biotinylated BSA conjugates bearing the synthetic peptides containing aa 3–20 of TYLCV CP (CP-NLS) or aa 84–106 of BDMV BV1 (BV1-NLS) were also imported into the nuclei of permeabilized cells. This import was blocked by the addition of unlabelled BSA–NLS peptide conjugates or excess unlabelled free NLS peptides. The CP- and BV1-NLS peptides also mediated nuclear import of fluorescently labelled BSA molecules into the nuclei of microinjected mesophyll cells of Nicotiana benthamiana leaves, demonstrating their biological function in intact plant tissue. BV1-NLS and CP-NLS were shown to mediate specific binding to importin α, both in vitro and in vivo. These results are consistent with a common nuclear-import pathway for CP and BV1, probably via importin α.

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Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme

Molecular Biology of the Cell ◽

10.1091/mbc.e10-05-0461 ◽

2011 ◽

Vol 22 (5) ◽

pp. 652-660 ◽

Cited By ~ 16

Author(s):

Marie Christine Moutty ◽

Volkan Sakin ◽

Frauke Melchior

Keyword(s):

Amino Acids ◽

Nuclear Localization ◽

Nuclear Import ◽

Enzyme Complex ◽

Intact Cells ◽

Nuclear Localization Signals ◽

Importin Α ◽

Essential Enzyme ◽

Nuclear Targets

SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

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Towards plant resistance to viruses using protein-only RNase P

Nature Communications ◽

10.1038/s41467-021-21338-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anthony Gobert ◽

Yifat Quan ◽

Mathilde Arrivé ◽

Florent Waltz ◽

Nathalie Da Silva ◽

...

Keyword(s):

Mosaic Virus ◽

Virus Replication ◽

Yield Loss ◽

Rnase P ◽

Plant Viruses ◽

Yellow Mosaic Virus ◽

Resistance To Viruses ◽

Target Plant

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

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Breeding for Enhancing Legumovirus Resistance in Mungbean: Current Understanding and Future Directions

Agronomy ◽

10.3390/agronomy9100622 ◽

2019 ◽

Vol 9 (10) ◽

pp. 622 ◽

Cited By ~ 1

Author(s):

Chandra Mohan Singh ◽

Poornima Singh ◽

Aditya Pratap ◽

Rakesh Pandey ◽

Shalini Purwar ◽

...

Keyword(s):

Mosaic Virus ◽

Seed Quality ◽

Soil Health ◽

Mosaic Disease ◽

Bipartite Begomoviruses ◽

Yellow Mosaic Virus ◽

Symbiotic Association ◽

Detection Techniques ◽

Vigna Species ◽

Mungbean Yellow Mosaic Virus

Yellow mosaic disease (YMD) affects several types of leguminous crops, including the Vigna species, which comprises a number of commercially important pulse crops. YMD is characterized by the formation of a bright yellow mosaic pattern on the leaves; in severe forms, this pattern can also be seen on stems and pods. This disease leads to tremendous yield losses, even up to 100%, in addition to deterioration in seed quality. Symptoms of this disease are similar among affected plants; YMD is not limited to mungbean (Vigna radiata L. Wilczek) and also affects other collateral and alternate hosts. In the last decade, rapid advancements in molecular detection techniques have been made, leading to an improved understanding of YMD-causing viruses. Three distinct bipartite begomoviruses, namely, Mungbean Yellow Mosaic India Virus (MYMIV), Mungbean Yellow Mosaic Virus (MYMV), and Horsegram Yellow Mosaic Virus (HgYMV), are known to cause YMD in Vigna spp. Vigna crops serve as an excellent protein source for vegetarians worldwide; moreover, they aid in improving soil health by fixing atmospheric nitrogen through a symbiotic association with Rhizobium bacteria. The loss in the yield of these short-duration crops due to YMD, thus, needs to be checked. This review highlights the discoveries that have been made regarding various aspects of YMD affecting mungbean, including the determination of YMD-causing viruses and strategies used to develop high-yielding YMD-resistant mungbean varieties that harness the potential of related Vigna species through the use of different omics approaches.

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