scholarly journals 3C protease of enterovirus 71 cleaves promyelocytic leukemia protein and impairs PML-NBs production

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Zhuoran Li ◽  
Ya’ni Wu ◽  
Hui Li ◽  
Wenqian Li ◽  
Juan Tan ◽  
...  
Keyword(s):  
Enterovirus 71 ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Ev71 Infection ◽  
3C Protease ◽  
Pml Nuclear Bodies ◽  
Antiviral Function ◽  
Hand Foot Mouth Disease ◽  
Antiviral Activities ◽  
Novel Target

Abstract Background Enterovirus 71 (EV71) usually infects infants causing hand-foot-mouth disease (HFMD), even fatal neurological disease like aseptic meningitis. Effective drug for preventing and treating EV71 infection is unavailable currently. EV71 3C mediated the cleavage of many proteins and played an important role in viral inhibiting host innate immunity. Promyelocytic leukemia (PML) protein, the primary organizer of PML nuclear bodies (PML-NBs), can be induced by interferon and is involved in antiviral activity. PML inhibits EV71 replication, and EV71 infection reduces PML expression, but the molecular mechanism is unclear. Methods The cleavage of PMLIII and IV was confirmed by co-transfection of EV71 3C protease and PML. The detailed cleavage sites were evaluated further by constructing the Q to A mutant of PML. PML knockout cells were infected with EV71 to identify the effect of cleavage on EV71 replication. Immunofluorescence analysis to examine the interference of EV71 3C on the formation of PML-NBs. Results EV71 3C directly cleaved PMLIII and IV. Furthermore, 3C cleaved PMLIV at the sites of Q430–A431 and Q444–S445 through its protease activity. Overexpression of PMLIV Q430A/Q444A variant exhibited stronger antiviral potential than the wild type. PMLIV Q430A/Q444A formed normal nuclear bodies that were not affected by 3C, suggesting that 3C may impair PML-NBs production via PMLIV cleavage and counter its antiviral activities. PML, especially PMLIV, which sequesters viral proteins in PML-NBs and inhibits viral production, is a novel target of EV71 3C cleavage. Conclusions EV71 3C cleaves PMLIV at Q430–A431 and Q444–S445. Cleavage reduces the antiviral function of PML and decomposes the formation of PML-NBs, which is conducive to virus replication.

scholarly journals Adenovirus E1B 55-Kilodalton Protein Is a p53-SUMO1 E3 Ligase That Represses p53 and Stimulates Its Nuclear Export through Interactions with Promyelocytic Leukemia Nuclear Bodies

2010 ◽  
Vol 84 (23) ◽  
pp. 12210-12225 ◽  
Author(s):  
Mario A. Pennella ◽  
Yue Liu ◽  
Jennifer L. Woo ◽  
Chongwoo A. Kim ◽  
Arnold J. Berk
Keyword(s):  
Nuclear Export ◽  
P53 Mutation ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Live Cells ◽  
High Concentration ◽  
Sumoylation Site ◽  
Pml Nuclear Bodies ◽  
Induced Apoptosis

ABSTRACT Oncogenic transformation by adenovirus E1A and E1B-55K requires E1B-55K inhibition of p53 activity to prevent E1A-induced apoptosis. During viral infection, E1B-55K and E4orf6 substitute for the substrate-binding subunits of the host cell cullin 5 class of ubiquitin ligases, resulting in p53 polyubiquitinylation and proteasomal degradation. Here we show that E1B-55K alone also functions as an E3 SUMO1-p53 ligase. Fluorescence microscopy studies showed that E1B-55K alone, in the absence of other viral proteins, causes p53 to colocalize with E1B-55K in promyelocytic leukemia (PML) nuclear bodies, nuclear domains with a high concentration of sumoylated proteins. Photobleaching experiments with live cells revealed that E1B-55K tethering of p53 in PML nuclear bodies decreases the in vivo nuclear mobility of p53 nearly 2 orders of magnitude. E1B-55K-induced p53 sumoylation contributes to maximal inhibition of p53 function since mutation of the major p53 sumoylation site decreases E1B-55K-induced p53 sumoylation, tethering in PML nuclear bodies, and E1B-55K inhibition of p53 activity. Mutation of the E1B-55K sumoylation site greatly inhibits E1B-55K association with PML nuclear bodies and the p53 nuclear export to cytoplasmic aggresomes observed in E1A-E1B-transformed cells. Purified E1B-55K and p53 form high-molecular-weight complexes potentially through the formation of a network of E1B-55K dimers bound to the N termini of p53 tetramers. In support of this model, a p53 mutation that prevents tetramer formation greatly reduces E1B-55K-induced tethering in PML nuclear bodies and p53 nuclear export. These data indicate that E1B-55K's association with PML nuclear bodies inactivates p53 by first sequestering it in PML nuclear bodies and then greatly facilitating its nuclear export.

scholarly journals Enterovirus 71 Represses Interleukin Enhancer-Binding Factor 2 Production and Nucleus Translocation to Antagonize ILF2 Antiviral Effects

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 22
Author(s):  
Jing Jin ◽  
Wenbiao Wang ◽  
Sha Ai ◽  
Weiyong Liu ◽  
Yu Song ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Enterovirus 71 ◽  
Ev71 Infection ◽  
Health It ◽  
Antiviral Effects ◽  
Distinct Mechanism ◽  
Binding Factor ◽  
2B Protein ◽  
Rd Cells ◽  
Hand Foot Mouth Disease

Enterovirus 71 (EV71) infection causes hand-foot-mouth disease (HFMD), meningoencephalitis, neonatal sepsis, and even fatal encephalitis in children, thereby presenting a serious risk to public health. It is important to determine the mechanisms underlying the regulation of EV71 infection. In this study, we initially show that the interleukin enhancer-binding factor 2 (ILF2) reduces EV71 50% tissue culture infective dose (TCID50) and attenuates EV71 plaque-formation unit (PFU), thereby repressing EV71 infection. Microarray data analyses show that ILF2 mRNA is reduced upon EV71 infection. Cellular studies indicate that EV71 infection represses ILF2 mRNA expression and protein production in human leukemic monocytes (THP-1) -differentiated macrophages and human rhabdomyosarcoma (RD) cells. In addition, EV71 nonstructural protein 2B interacts with ILF2 in human embryonic kidney (HEK293T) cells. Interestingly, in the presence of EV71 2B, ILF2 is translocated from the nucleus to the cytoplasm, and it colocalizes with 2B in the cytoplasm. Therefore, we present a distinct mechanism by which EV71 antagonizes ILF2-mediated antiviral effects by inhibiting ILF2 expression and promoting ILF2 translocation from the nucleus to the cytoplasm through its 2B protein.

scholarly journals Plant-Produced Anti-Enterovirus 71 (EV71) Monoclonal Antibody Efficiently Protects Mice Against EV71 Infection

Plants ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 560 ◽  
Author(s):  
Kaewta Rattanapisit ◽  
Zhang Chao ◽  
Konlavat Siriwattananon ◽  
Zhong Huang ◽  
Waranyoo Phoolcharoen
Keyword(s):  
Monoclonal Antibody ◽  
Therapeutic Option ◽  
Enterovirus 71 ◽  
Cost Effective ◽  
Ev71 Infection ◽  
Asia Pacific ◽  
Variable Regions ◽  
Hand Foot Mouth Disease ◽  
Protection Against Infection

Enterovirus 71 (EV71) is the main causative agent of severe hand-foot-mouth disease. EV71 affects countries mainly in the Asia-Pacific region, which makes it unattractive for pharmaceutical companies to develop drugs or vaccine to combat EV71 infection. However, development of these drugs and vaccines is vital to protect younger generations. This study aims to develop a specific monoclonal antibody (mAb) to EV71 using a plant platform, which is a cost-effective and scalable production technology. A previous report showed that D5, a murine anti-EV71 mAb, binds to VP1 protein of EV71, potently neutralizes EV71 in vitro, and effectively protects mice against EV71 infection. Herein, plant-produced chimeric D5 (cD5) mAb, variable regions of murine D5 antibody linked with constant regions of human IgG1, was transiently expressed in Nicotiana benthamiana using geminiviral vectors. The antibody was expressed at high levels within six days of infiltration. Plant-produced cD5 retained its in vitro high-affinity binding and neutralizing activity against EV71. Furthermore, a single dose (10 µg/g body weight) of plant-produced cD5 mAb offered 100% protection against infection in mice after a lethal EV71 challenge. Therefore, our results showed that plant-produced anti-EV71 mAb is an effective, safe, and affordable therapeutic option against EV71 infection.

scholarly journals Immunocytochemical Diagnosis of Acute Promyelocytic Leukemia (M3) With the Monoclonal Antibody PG-M3 (Anti-PML)

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 4046-4053 ◽  
Author(s):  
Brunangelo Falini ◽  
Leonardo Flenghi ◽  
Marta Fagioli ◽  
Francesco Lo Coco ◽  
Iole Cordone ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Acute Promyelocytic Leukemia ◽  
Detection System ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Nuclear Staining ◽  
Wild Type ◽  
Amino Terminal ◽  
Pml Nuclear Bodies

Abstract Acute promyelocytic leukemia (APL) is characterized by a reciprocal 15; 17 chromosomal translocation, which fuses the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes, leading to the expression of the PML/RARα fusion oncoprotein. Immunocytochemical labeling of the wild-type PML protein with the PG-M3 monoclonal antibody (MoAb) directed against the amino terminal portion of the human PML gene product, produces a characteristic nuclear speckled pattern that is due to localization of the protein into discrete dots (5 to 20 per nucleus), named PML nuclear bodies. The architecture of PML nuclear bodies appears to be disrupted in APL cells that bear the t(15; 17), thus resulting in a change of the nuclear staining pattern from speckled (wild-type PML protein) to microgranular (PML-RARα fusion protein). To assess whether the PG-M3 MoAb could assist in the diagnosis of APL (M3), bone marrow and/or peripheral blood samples from 100 cases of acute nonlymphoid leukemias of different subtypes were blindly immunostained with the PG-M3 MoAb, using the immunoalkaline phosphatase (APAAP) or immunofluorescence technique as detection system. Notably, the abnormal (micropunctate) pattern of the PML/RARα fusion protein (usually ≥50 small granules/per nucleus) was observed in APL (M3) samples, but not in other types of acute nonlymphoid leukemias. Immunocytochemical labeling with PG-M3 was particularly useful in the diagnosis of microgranular variant of APL (M3V) (three cases misdiagnosed as M4 and M5), and also to exclude a morphologic misdiagnosis of APL (six of 78 cases). In all cases investigated, immunocytochemical results were in agreement with those of reverse transcription-polymerase chain reaction (RT-PCR) for PML/RARα. Because the epitope identified by PG-M3 is located in the aminoterminal portion of PML (AA 37 to 51), the antibody was suitable for recognizing APL cases characterized by breakpoint occurring at different sites of PML (bcr 1, bcr 2 and bcr 3). In conclusion, immunocytochemical labeling with PG-M3 represents a rapid, sensitive, and highly-specific test for the diagnosis of APL that bears the t(15; 17). This should allow an easy and correct diagnosis of this subtype of acute leukemia to any laboratory provided with a minimal equipment for immunocytochemistry work.

scholarly journals Nuclear and cytoplasmic shuttling of TRADD induces apoptosis via different mechanisms

2002 ◽  
Vol 157 (6) ◽  
pp. 975-984 ◽  
Author(s):  
Michael Morgan ◽  
Jacqueline Thorburn ◽  
Pier Paolo Pandolfi ◽  
Andrew Thorburn
Keyword(s):  
Nuclear Export ◽  
Tumor Necrosis Factor Receptor ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Nuclear Bodies ◽  
Death Domain ◽  
Apoptosis Pathway ◽  
Promyelocytic Leukemia Protein ◽  
Caspase Inhibitors ◽  
Pml Nuclear Bodies

The adapter protein tumor necrosis factor receptor (TNFR)1–associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.

scholarly journals Arsenic-Induced SUMO-Dependent Recruitment of RNF4 into PML Nuclear Bodies

2010 ◽  
Vol 21 (23) ◽  
pp. 4227-4239 ◽  
Author(s):  
Marie-Claude Geoffroy ◽  
Ellis G. Jaffray ◽  
Katherine J. Walker ◽  
Ronald T. Hay
Keyword(s):  
Retinoic Acid Receptor ◽  
E3 Ligase ◽  
Nuclear Body ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Dependent Manner ◽  
Nuclear Trafficking ◽  
Sumo Modification ◽  
Ubiquitin E3 Ligase ◽  
Pml Nuclear Bodies

In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML.

scholarly journals PML is a ROS sensor activating p53 upon oxidative stress

2017 ◽  
Vol 214 (11) ◽  
pp. 3197-3206 ◽  
Author(s):  
Michiko Niwa-Kawakita ◽  
Omar Ferhi ◽  
Hassane Soilihi ◽  
Morgane Le Bras ◽  
Valérie Lallemand-Breitenbach ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Biology ◽  
Antioxidant Properties ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Glutathione Depletion ◽  
Pml Nuclear Bodies ◽  
P53 Activation ◽  
Induced Changes

Promyelocytic leukemia (PML) nuclear bodies (NBs) recruit partner proteins, including p53 and its regulators, thereby controlling their abundance or function. Investigating arsenic sensitivity of acute promyelocytic leukemia, we proposed that PML oxidation promotes NB biogenesis. However, physiological links between PML and oxidative stress response in vivo remain unexplored. Here, we identify PML as a reactive oxygen species (ROS) sensor. Pml−/− cells accumulate ROS, whereas PML expression decreases ROS levels. Unexpectedly, Pml−/− embryos survive acute glutathione depletion. Moreover, Pml−/− animals are resistant to acetaminophen hepatotoxicity or fasting-induced steatosis. Molecularly, Pml−/− animals fail to properly activate oxidative stress–responsive p53 targets, whereas the NRF2 response is amplified and accelerated. Finally, in an oxidative stress–prone background, Pml−/− animals display a longevity phenotype, likely reflecting decreased basal p53 activation. Thus, similar to p53, PML exerts basal antioxidant properties but also drives oxidative stress–induced changes in cell survival/proliferation or metabolism in vivo. Through NB biogenesis, PML therefore couples ROS sensing to p53 responses, shedding a new light on the role of PML in senescence or stem cell biology.

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