scholarly journals Geminivirus Replication Protein impairs SUMO conjugation of PCNA at two acceptor sites

2018 ◽  
Author(s):  
Manuel Arroyo-Mateos ◽  
Blanca Sabarit ◽  
Francesca Maio ◽  
Miguel A. Sánchez-Durán ◽  
Tabata Rosas-Díaz ◽  
...  
Keyword(s):  
Dna Repair ◽  
Infected Plant ◽  
Plant Cells ◽  
Nuclear Antigen ◽  
Replication Machinery ◽  
In Planta ◽  
Post Translational Modification ◽  
Dna Viruses ◽  
Multifunctional Protein ◽  
Sumo Conjugation

ABSTRACTGeminiviruses are DNA viruses that replicate in nuclei of infected plant cells using the plant DNA replication machinery, including PCNA (Proliferating cellular nuclear antigen), a cofactor that orchestrates genome duplication and maintenance by recruiting crucial players to replication forks. These viruses encode a multifunctional protein, Rep, which is essential for viral replication, induces the accumulation of the host replication machinery and interacts with several host proteins, including PCNA and the SUMO E2 conjugation enzyme (SCE1). Post-translational modification of PCNA by ubiquitin or SUMO plays an essential role in the switching of PCNA between interacting partners during DNA metabolism processes (e.g. replication, recombination, repair, etc.). In yeast, PCNA sumoylation has been associated to DNA repair involving homologous recombination (HR). Previously, we reported that ectopic Rep expression results in very specific changes in the sumoylation pattern of plant cells. In this work, we show, using a reconstituted sumoylation system inEscherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that co-expression of the geminivirus protein Rep suppresses sumoylation at these lysines. Finally, we confirm that PCNA is sumoylatedin plantaand that Rep also interferes with PCNA sumoylation in plant cells.ImportanceSUMO adducts have a key role in regulating the activity of animal and yeast PCNA on DNA repair and replication. Our work demonstrates for the first time that sumoylation of plant PCNA occurs in plant cells and that a plant virus interferes with this modification. This work marks the importance of sumoylation in allowing viral infection and replication in plants. Moreover, it constitutes a prime example of viral proteins interfering with post-translational modifications of selected host factors to create a proper environment for infection.

scholarly journals Geminivirus Replication Protein Impairs SUMO Conjugation of Proliferating Cellular Nuclear Antigen at Two Acceptor Sites

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Manuel Arroyo-Mateos ◽  
Blanca Sabarit ◽  
Francesca Maio ◽  
Miguel A. Sánchez-Durán ◽  
Tabata Rosas-Díaz ◽  
...  
Keyword(s):  
Dna Repair ◽  
Posttranslational Modifications ◽  
Infected Plant ◽  
Plant Cells ◽  
Nuclear Antigen ◽  
Replication Machinery ◽  
In Planta ◽  
Content Type ◽  
Multifunctional Protein ◽  
Cellular Nuclear Antigen

ABSTRACTGeminiviruses are DNA viruses that replicate in nuclei of infected plant cells using the plant DNA replication machinery, including PCNA (proliferating cellular nuclear antigen), a cofactor that orchestrates genome duplication and maintenance by recruiting crucial players to replication forks. These viruses encode a multifunctional protein, Rep, which is essential for viral replication, induces the accumulation of the host replication machinery, and interacts with several host proteins, including PCNA and the SUMO E2 conjugation enzyme (SCE1). Posttranslational modification of PCNA by ubiquitin or SUMO plays an essential role in the switching of PCNA between interacting partners during DNA metabolism processes (e.g., replication, recombination, and repair, etc.). In yeast, PCNA sumoylation has been associated with DNA repair involving homologous recombination (HR). Previously, we reported that ectopic Rep expression results in very specific changes in the sumoylation pattern of plant cells. In this work, we show, using a reconstituted sumoylation system inEscherichia coli, that tomato PCNA is sumoylated at two residues, K254 and K164, and that coexpression of the geminivirus protein Rep suppresses sumoylation at these lysines. Finally, we confirm that PCNA is sumoylatedin plantaand that Rep also interferes with PCNA sumoylation in plant cells.IMPORTANCESUMO adducts have a key role in regulating the activity of animal and yeast PCNA on DNA repair and replication. Our work demonstrates for the first time that sumoylation of plant PCNA occurs in plant cells and that a plant virus interferes with this modification. This work marks the importance of sumoylation in allowing viral infection and replication in plants. Moreover, it constitutes a prime example of how viral proteins interfere with posttranslational modifications of selected host factors to create a proper environment for infection.

scholarly journals CRISPR-Cas13d mediates robust RNA virus interference in plants

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Ahmed Mahas ◽  
Rashid Aman ◽  
Magdy Mahfouz
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Viruses ◽  
Rna Virus ◽  
Plant Cells ◽  
In Planta ◽  
Dna Viruses ◽  
Archaeal Species ◽  
Target Rna ◽  
Virus Biology ◽  
Key Questions

Abstract Background CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana. Results Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses simultaneously, with robust interference efficiencies. In addition, CasRx exhibits strong specificity against the target virus and does not exhibit collateral activity in planta. Conclusions Our data establish CasRx as the most robust Cas13 variant for RNA virus interference applications in planta and demonstrate its suitability for studying key questions relating to virus biology.

scholarly journals Interaction between a Geminivirus Replication Protein and the Plant Sumoylation System

2004 ◽  
Vol 78 (6) ◽  
pp. 2758-2769 ◽  
Author(s):  
A. G. Castillo ◽  
L. J. Kong ◽  
L. Hanley-Bowdoin ◽  
E. R. Bejarano
Keyword(s):  
Protein A ◽  
Infected Plant ◽  
Replication Machinery ◽  
Host Proteins ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
Oligomeric Protein ◽  
Essential Components ◽  
Nuclear Cell ◽  
Two Hybrid

ABSTRACT Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells after accumulation of host replication machinery. Tomato golden mosaic virus (TGMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) encode a protein, RepAC1 (or Rep), that is essential for viral replication. Rep/RepAC1 is an oligomeric protein that binds to double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and is sufficient for host induction. It also interacts with several host proteins, including the cell cycle regulator, retinoblastoma, and essential components of the cell DNA replication machinery, like proliferating nuclear cell antigen (PCNA) and RFC-1. To identify other cellular proteins that interact with Rep/RepAC1 protein, a Nicotiana benthamiana cDNA library was screened with a yeast two-hybrid assay. The host cell sumoylation enzyme, NbSCE1 (N. benthamiana SUMO-conjugating enzyme, homolog to Saccharomyces cerevisiae UBC9), was found to interact specifically with RepAC1. Mapping studies localized the interaction to the N-terminal half of RepAC1. Effects on geminivirus replication were observed in transgenic plants with altered levels of SUMO, the substrate for UBC9.

scholarly journals SUMO, a small, but powerful, regulator of double-strand break repair

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160281 ◽  
Author(s):  
Alexander J. Garvin ◽  
Joanna R. Morris
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Repair Process ◽  
Strand Break ◽  
Post Translational Modification ◽  
Link Type ◽  
Sumo Conjugation ◽  
Dna Lesion ◽  
Sumo Proteases

The response to a DNA double-stranded break in mammalian cells is a process of sensing and signalling the lesion. It results in halting the cell cycle and local transcription and in the mediation of the DNA repair process itself. The response is launched through a series of post-translational modification signalling events coordinated by phosphorylation and ubiquitination. More recently modifications of proteins by S mall U biquitin-like MO difier (SUMO) isoforms have also been found to be key to coordination of the response (Morris et al. 2009 Nature 462 , 886–890 ( doi:10.1038/nature08593 ); Galanty et al. 2009 Nature 462 , 935–939 ( doi:10.1038/nature08657 )). However our understanding of the role of SUMOylation is slight compared with our growing knowledge of how ubiquitin drives signal amplification and key chromatin interactions. In this review we consider our current knowledge of how SUMO isoforms, SUMO conjugation machinery, SUMO proteases and SUMO-interacting proteins contribute to directing altered chromatin states and to repair-protein kinetics at a double-stranded DNA lesion in mammalian cells. We also consider the gaps in our understanding. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

scholarly journals Potential Phosphorylation of Viral Nonstructural Protein 1 in Dengue Virus Infection

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1393
Author(s):  
Thanyaporn Dechtawewat ◽  
Sittiruk Roytrakul ◽  
Yodying Yingchutrakul ◽  
Sawanya Charoenlappanit ◽  
Bunpote Siridechadilok ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Denv Infection ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modification ◽  
Multifunctional Protein ◽  
Nonstructural Protein 1 ◽  
Underlying Mechanisms

Dengue virus (DENV) infection causes a spectrum of dengue diseases that have unclear underlying mechanisms. Nonstructural protein 1 (NS1) is a multifunctional protein of DENV that is involved in DENV infection and dengue pathogenesis. This study investigated the potential post-translational modification of DENV NS1 by phosphorylation following DENV infection. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), 24 potential phosphorylation sites were identified in both cell-associated and extracellular NS1 proteins from three different cell lines infected with DENV. Cell-free kinase assays also demonstrated kinase activity in purified preparations of DENV NS1 proteins. Further studies were conducted to determine the roles of specific phosphorylation sites on NS1 proteins by site-directed mutagenesis with alanine substitution. The T27A and Y32A mutations had a deleterious effect on DENV infectivity. The T29A, T230A, and S233A mutations significantly decreased the production of infectious DENV but did not affect relative levels of intracellular DENV NS1 expression or NS1 secretion. Only the T230A mutation led to a significant reduction of detectable DENV NS1 dimers in virus-infected cells; however, none of the mutations interfered with DENV NS1 oligomeric formation. These findings highlight the importance of DENV NS1 phosphorylation that may pave the way for future target-specific antiviral drug design.

scholarly journals Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

2021 ◽  
Vol 9 (2) ◽  
pp. 295
Author(s):  
Yvann Bourigault ◽  
Andrea Chane ◽  
Corinne Barbey ◽  
Sylwia Jafra ◽  
Robert Czajkowski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Infected Plant ◽  
Soft Rot ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Cell Physiology ◽  
In Planta ◽  
Reporter System ◽  
Cell Wall Lysis

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

scholarly journals Highly Specialized Ubiquitin-Like Modifications: Shedding Light into the UFM1 Enigma

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 255
Author(s):  
Katharina F. Witting ◽  
Monique P.C. Mulder
Keyword(s):  
Dna Repair ◽  
Stress Response ◽  
Embryonic Development ◽  
Er Stress ◽  
Unmet Needs ◽  
Biological Function ◽  
Post Translational Modification ◽  
Er Stress Response ◽  
Cellular Events ◽  
Complex Signaling

Post-translational modification with Ubiquitin-like proteins represents a complex signaling language regulating virtually every cellular process. Among these post-translational modifiers is Ubiquitin-fold modifier (UFM1), which is covalently attached to its substrates through the orchestrated action of a dedicated enzymatic cascade. Originally identified to be involved embryonic development, its biological function remains enigmatic. Recent research reveals that UFM1 regulates a variety of cellular events ranging from DNA repair to autophagy and ER stress response implicating its involvement in a variety of diseases. Given the contribution of UFM1 to numerous pathologies, the enzymes of the UFM1 cascade represent attractive targets for pharmacological inhibition. Here we discuss the current understanding of this cryptic post-translational modification especially its contribution to disease as well as expand on the unmet needs of developing chemical and biochemical tools to dissect its role.

scholarly journals ROS induces NETosis by oxidizing DNA and initiating DNA repair

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Dhia Azzouz ◽  
Meraj A. Khan ◽  
Nades Palaniyar
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Polymerase Activity ◽  
Neutrophil Activation ◽  
Neutrophil Extracellular Trap ◽  
Nuclear Antigen ◽  
Chromatin Decondensation ◽  
Genome Wide ◽  
Repair Protein ◽  
Dna Repair Protein

AbstractReactive oxygen species (ROS) are essential for neutrophil extracellular trap (NET) formation or NETosis. Nevertheless, how ROS induces NETosis is unknown. Neutrophil activation induces excess ROS production and a meaningless genome-wide transcription to facilitate chromatin decondensation. Here we show that the induction of NADPH oxidase-dependent NETosis leads to extensive DNA damage, and the subsequent translocation of proliferating cell nuclear antigen (PCNA), a key DNA repair protein, stored in the cytoplasm into the nucleus. During the activation of NETosis (e.g., by phorbol myristate acetate, Escherichia coli LPS, Staphylococcus aureus (RN4220), or Pseudomonas aeruginosa), preventing the DNA-repair-complex assembly leading to nick formation that decondenses chromatin causes the suppression of NETosis (e.g., by inhibitors to, or knockdown of, Apurinic endonuclease APE1, poly ADP ribose polymerase PARP, and DNA ligase). The remaining repair steps involving polymerase activity and PCNA interactions with DNA polymerases β/δ do not suppress agonist-induced NETosis. Therefore, excess ROS produced during neutrophil activation induces NETosis by inducing extensive DNA damage (e.g., oxidising guanine to 8-oxoguanine), and the subsequent DNA repair pathway, leading to chromatin decondensation.

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