Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target

ABSTRACTSARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and a PLA assay. In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase of PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

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Maneuvers on PCNA Rings during DNA Replication and Repair

Genes ◽

10.3390/genes9080416 ◽

2018 ◽

Vol 9 (8) ◽

pp. 416 ◽

Cited By ~ 23

Author(s):

Dea Slade

Keyword(s):

Dna Replication ◽

Protein Interactions ◽

Proliferating Cell Nuclear Antigen ◽

Binding Proteins ◽

Nuclear Antigen ◽

Vast Number ◽

Post Translational Modifications ◽

Dna Replication And Repair ◽

Replicative Polymerases ◽

Proliferating Cell

DNA replication and repair are essential cellular processes that ensure genome duplication and safeguard the genome from deleterious mutations. Both processes utilize an abundance of enzymatic functions that need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair at replication forks. Post-translational modifications of PCNA, ubiquitination and acetylation in particular, regulate the dynamics of PCNA-protein interactions. Proliferating cell nuclear antigen (PCNA) monoubiquitination elicits ‘polymerase switching’, whereby stalled replicative polymerase is replaced with a specialized polymerase, while PCNA acetylation may reduce the processivity of replicative polymerases to promote homologous recombination-dependent repair. While regulatory functions of PCNA ubiquitination and acetylation have been well established, the regulation of PCNA-binding proteins remains underexplored. Considering the vast number of PCNA-binding proteins, many of which have similar PCNA binding affinities, the question arises as to the regulation of the strength and sequence of their binding to PCNA. Here I provide an overview of post-translational modifications on both PCNA and PCNA-interacting proteins and discuss their relevance for the regulation of the dynamic processes of DNA replication and repair.

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An evaluation of antioxidant effects on recovery from postischemic acute renal failure.

Journal of the American Society of Nephrology ◽

10.1681/asn.v481588 ◽

1994 ◽

Vol 4 (8) ◽

pp. 1588-1597

Author(s):

R A Zager ◽

S M Fuerstenberg ◽

P H Baehr ◽

D Myerson ◽

B Torok-Storb

Keyword(s):

Renal Failure ◽

Acute Renal Failure ◽

Dna Fragmentation ◽

Proliferating Cell Nuclear Antigen ◽

Antigen Expression ◽

Nuclear Antigen ◽

Terminal Deoxynucleotidyl Transferase ◽

Tubular Necrosis ◽

Proliferating Cell

Xanthine oxidase (XO) activity and hydroxyl radical (.OH) formation are widely proposed mediators of renal reperfusion injury, potentially altering the severity of, and recovery from, postischemic acute renal failure. The goal of this study was to ascertain whether combination XO inhibitor (oxypurinol) and .OH scavenger (Na benzoate) therapy, given at the time of renal ischemia, alters the extent of: (1) tubular necrosis and filtration failure; (2) DNA fragmentation/apoptosis (assessed in situ by terminal deoxynucleotidyl transferase reactivity); (3) early tubular regenerative responses (proliferating cell nuclear antigen expression; (3H)thymidine incorporation); and (4) the rate and/or degree of functional and morphologic repair. The effects of XO inhibition, .OH scavengers, and "catalytic" iron (FeSO4) on human proximal tubular cell proliferation in vitro were also assessed with a newly established cell line (HK-2). Male Sprague-Dawley rats were subjected to 35 min of bilateral renal arterial occlusion with or without oxypurinol/benzoate therapy. These agents did not alter the extent of tubular necrosis or filtration failure, proliferating cell nuclear antigen expression or thymidine incorporation, or the rate/extent of renal functional/morphologic repair. DNA fragmentation did not precede tubular necrosis, and it was unaffected by antioxidant therapy. By 5 days postischemia, both treatment groups demonstrated regenerating epithelial fronds that protruded into the lumina. These structures contained terminal deoxynucleotidyl transferase-reactive, but morphologically intact, cells, suggesting the presence of apoptosis. Oxypurinol and .OH scavengers (benzoate; dimethylthiourea) suppressed in vitro tubular cell proliferation; conversely, catalytic Fe had a growth-stimulatory effect. These results suggest that: (1) XO inhibition/.OH scavenger therapy has no discernible net effect on postischemic acute renal failure; (2) DNA fragmentation does not precede tubular necrosis, suggesting that it is not a primary mediator of ischemic cell death; and (3) antioxidants can be antiproliferative for human tubular cells, possibly mitigating their potential beneficial effects.

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Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.57-66.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 57-66

Author(s):

M Zuber ◽

E M Tan ◽

M Ryoji

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Living Cells ◽

Nuclear Antigen ◽

Plasmid Replication ◽

Dna Polymerase Delta ◽

Dna Polymerase Alpha ◽

Proliferating Cell

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

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Oxidative DNA Damage Bypass in Arabidopsis thaliana Requires DNA Polymerase λ and Proliferating Cell Nuclear Antigen 2

The Plant Cell ◽

10.1105/tpc.110.081455 ◽

2011 ◽

Vol 23 (2) ◽

pp. 806-822 ◽

Cited By ~ 36

Author(s):

Alessandra Amoroso ◽

Lorenzo Concia ◽

Caterina Maggio ◽

Cécile Raynaud ◽

Catherine Bergounioux ◽

...

Keyword(s):

Dna Damage ◽

Arabidopsis Thaliana ◽

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Oxidative Dna Damage ◽

Nuclear Antigen ◽

Dna Polymerase Λ ◽

Proliferating Cell

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TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004122117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23588-23596

Author(s):

Min Li ◽

Xiaohua Xu ◽

Chou-Wei Chang ◽

Yilun Liu

Keyword(s):

Rna Polymerase Ii ◽

Proliferating Cell Nuclear Antigen ◽

E3 Ligase ◽

Nuclear Antigen ◽

Dna Breaks ◽

Interacting Protein ◽

Proteomic Approach ◽

Sumo E3 Ligase ◽

Proliferating Cell

In human cells, the DNA replication factor proliferating cell nuclear antigen (PCNA) can be conjugated to either the small ubiquitinlike modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is induced by transcription to facilitate resolution of transcription–replication conflict (TRC). To date, the SUMO E3 ligase that provides substrate specificity for SUMO2-PCNA conjugation in response to TRC remains unknown. Using a proteomic approach, we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA conjugation. In vitro, TRIM28, together with the RNA polymerase II (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA conjugation but inhibits SUMO1-PCNA formation. This activity requires a PCNA-interacting protein (PIP) motif located within the bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA on human chromatin is dependent on both transcription and RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. As a consequence, TRIM28 depletion led to RNAPII accumulation at TRC sites, and expression of a TRIM28 PIP mutant failed to suppress TRC-induced DNA breaks.

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Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival

Journal of Experimental Medicine ◽

10.1084/jem.20092241 ◽

2010 ◽

Vol 207 (12) ◽

pp. 2631-2645 ◽

Cited By ~ 95

Author(s):

Véronique Witko-Sarsat ◽

Julie Mocek ◽

Dikra Bouayad ◽

Nicola Tamassia ◽

Jean-Antoine Ribeil ◽

...

Keyword(s):

Proliferating Cell Nuclear Antigen ◽

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Nuclear Antigen ◽

Neutrophil Apoptosis ◽

Proliferating Cells ◽

Rna Transfection ◽

Neutrophil Survival ◽

Proliferating Cell

Neutrophil apoptosis is a highly regulated process essential for inflammation resolution, the molecular mechanisms of which are only partially elucidated. In this study, we describe a survival pathway controlled by proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repairing of proliferating cells. We show that mature neutrophils, despite their inability to proliferate, express high levels of PCNA exclusively in their cytosol and constitutively associated with procaspases, presumably to prevent their activation. Notably, cytosolic PCNA abundance decreased during apoptosis, and increased during in vitro and in vivo exposure to the survival factor granulocyte colony-stimulating factor (G-CSF). Peptides derived from the cyclin-dependent kinase inhibitor p21, which compete with procaspases to bind PCNA, triggered neutrophil apoptosis thus demonstrating that specific modification of PCNA protein interactions affects neutrophil survival. Furthermore, PCNA overexpression rendered neutrophil-differentiated PLB985 myeloid cells significantly more resistant to TNF-related apoptosis-inducing ligand– or gliotoxin-induced apoptosis. Conversely, a decrease in PCNA expression after PCNA small interfering RNA transfection sensitized these cells to apoptosis. Finally, a mutation in the PCNA interdomain-connecting loop, the binding site for many partners, significantly decreased the PCNA-mediated antiapoptotic effect. These results identify PCNA as a regulator of neutrophil lifespan, thereby highlighting a novel target to potentially modulate pathological inflammation.

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Proliferating cell nuclear antigen destabilizes c-Abl tyrosine kinase and regulates cell apoptosis in response to DNA damage

APOPTOSIS ◽

10.1007/s10495-009-0313-2 ◽

2009 ◽

Vol 14 (3) ◽

pp. 268-275 ◽

Cited By ~ 9

Author(s):

Xiang He ◽

Congwen Wei ◽

Ting Song ◽

Jing Yuan ◽

Yanhong Zhang ◽

...

Keyword(s):

Dna Damage ◽

Tyrosine Kinase ◽

Cell Apoptosis ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Abl Tyrosine Kinase ◽

Proliferating Cell

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Proliferating cells in human eccrine and apocrine sweat glands.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.12.8537637 ◽

1995 ◽

Vol 43 (12) ◽

pp. 1217-1221 ◽

Cited By ~ 13

Author(s):

Y Morimoto ◽

K Saga

Keyword(s):

Proliferating Cell Nuclear Antigen ◽

Myoepithelial Cells ◽

Nuclear Antigen ◽

Sweat Glands ◽

Secretory Cells ◽

Proliferating Cells ◽

Peripheral Cells ◽

Proliferating Cell ◽

Eccrine Sweat Glands

Morphological observations of sweat glands showed degenerated debris of secretory cells in the secretory lumen in both apocrine and eccrine sweat glands. This suggested that dead secretory cells of human eccrine and apocrine sweat glands were released into the lumen and replaced by other cells. However, we did not know which type of cells replaced lost secretory cells. Therefore, we studied the proliferating cells in human eccrine and apocrine sweat glands by labeling S-phase cells in vitro with 5-bromo-2'-deoxyuridine (BrdUrd) and by immunostaining proliferation-associated proliferating cell nuclear antigen (PCNA) with anti-PCNA monoclonal antibody. BrdUrd and anti-PCNA antibody labeled a few secretory cells in eccrine and apocrine sweat glands, but neither method labeled myoepithelial cells. Luminal and peripheral cells of the eccrine and apocrine coiled duct were labeled with both BrdUrd and PCNA. However, we could not find any highly proliferative germinative cells in coiled ducts. Our results suggest that lost secretory cells could be replaced by proliferation of secretory cells themselves rather than by proliferation of myoepithelial cells or duct cells.

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Interaction between proliferating cell nuclear antigen (PCNA) and a DnaJ induced by DNA damage

Journal of Plant Research ◽

10.1007/s10265-005-0197-3 ◽

2005 ◽

Vol 118 (2) ◽

pp. 91-97 ◽

Cited By ~ 21

Author(s):

Taichi Yamamoto ◽

Yoko Mori ◽

Toyotaka Ishibashi ◽

Yukinobu Uchiyama ◽

Tadamasa Ueda ◽

...

Keyword(s):

Dna Damage ◽

Proliferating Cell Nuclear Antigen ◽

Nuclear Antigen ◽

Proliferating Cell

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Primer-DNA formation during simian virus 40 DNA replication in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2882 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2882-2890 ◽

Cited By ~ 18

Author(s):

D Denis ◽

P A Bullock

Keyword(s):

Dna Replication ◽

Proliferating Cell Nuclear Antigen ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Nuclear Antigen ◽

Origin Region ◽

Sv40 Dna ◽

Proliferating Cell

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.

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