scholarly journals UL36 Encoded by Marek’s Disease Virus Exhibits Linkage-Specific Deubiquitinase Activity

2020 ◽  
Vol 21 (5) ◽  
pp. 1783
Author(s):  
Junyan Lin ◽  
Yongxing Ai ◽  
Hongda Zhou ◽  
Yan Lv ◽  
Menghan Wang ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Protease Inhibitor ◽  
High Throughput Screening ◽  
Marek's Disease ◽  
Marek’S Disease ◽  
Wild Type ◽  
Ubiquitin Chain ◽  
Protein Ubiquitination ◽  
High Level

(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the Marek’s disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480)H234A and UL36(480)D232A, but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.

scholarly journals Herpesvirus of turkey reconstituted from bacterial artificial chromosome clones induces protection against Marek's disease

2006 ◽  
Vol 87 (4) ◽  
pp. 769-776 ◽  
Author(s):  
Susan J. Baigent ◽  
Lawrence J. Petherbridge ◽  
Lorraine P. Smith ◽  
Yuguang Zhao ◽  
Peter M. Chesters ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Artificial Chromosome ◽  
Marek's Disease ◽  
Wild Type Virus ◽  
Antigenic Relationship ◽  
Type Virus ◽  
Marek’S Disease ◽  
Wild Type ◽  
Bac Clones

Herpesvirus of turkey (HVT) is an alphaherpesvirus that is widely used as a live vaccine against Marek's disease because of its antigenic relationship with Marek's disease virus (MDV). In spite of a similar genome structure, HVT has several unique genes, the functions of which are not completely understood. As a first step in carrying out detailed analysis of the functions of the HVT genes, a full-length infectious bacterial artificial chromosome (BAC) clone of HVT was constructed. DNA from two independent BAC clones, upon transfection into chicken embryo fibroblasts, produced plaques similar to those produced by the wild-type virus. Viruses derived from the BAC clones were stable during in vitro passage, but showed differences in in vitro growth kinetics compared with the wild-type virus. Using a one-step mutagenesis protocol to delete the essential glycoprotein B gene from the HVT genome, followed by construction of the revertant virus, BAC clones of HVT were shown to be amenable to standard mutagenesis techniques. In spite of the difference in in vitro growth, viruses from both clones induced 100 % protection against infection by the virulent MDV strain RB-1B, indicating that the BAC-derived viruses could be used as vaccines with efficacies similar to that of the parental virus. The construction of HVT BAC is a major step in understanding the functions of HVT genes by exploiting the power of BAC technology. Furthermore, the availability of the BAC clones enables use of HVT as a vector for expressing foreign genes.

scholarly journals In Vitro Replication of Recombinant Marek’S Disease Viruses Constructed from Field Strains Lacking Meq and Vtr Oncogenes

2013 ◽  
Vol 57 (2) ◽  
pp. 141-147 ◽  
Author(s):  
Grzegorz Woźniakowski ◽  
Elżbieta Samorek-Salamonowicz
Keyword(s):  
Artificial Chromosome ◽  
Immunofluorescence Assay ◽  
Marek's Disease ◽  
Marek’S Disease ◽  
Wild Type ◽  
E Coli ◽  
Bac Clones ◽  
Potential Use ◽  
Chicken Embryo Fibroblasts

Abstract The study describes construction of five recombinant very virulent (vv) and very virulent plus (vv+) strains lacking meq and viral telomerase (vTR). Deletion of both copies of meq and vTR was achieved by Red E/T recombination in GS1783 E. coli cells. The constructed bacterial artificial chromosome (BAC) clones reconstituted in chicken embryo fibroblasts were examined by immunofluorescence assay to compare the features of recombinant strains with wild-type viruses. The results demonstrated that recombinant BAC strains caused slightly reduced cytophatic effect and decreased level of the fluorescence obtained from the monoclonal antibody in comparison to the parental viruses. Generation of recombinant BAC clones may provide more detailed information on the function of Marek's disease virus oncogenes and the potential use of recombinants for the preparation of the new vaccine against Marek’s disease.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals Rv3131, a gene encoding nitroreductase, is essential for metronidazole activation in Mycobacterium tuberculosis under hypoxic condition

2020 ◽  
Author(s):  
Wenzhu Dong ◽  
Jin Shi ◽  
Ping Chu ◽  
Rongmei Liu ◽  
Shu’an Wen ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Anaerobic Bacteria ◽  
Aerobic Condition ◽  
Hypoxic Condition ◽  
Significant Inhibition ◽  
Wild Type ◽  
Minimal Inhibitory Concentrations ◽  
High Level ◽  
Nitroreductase Activity

Abstract ObjectivesThe impressive potency of metronidazole (MTZ) against anaerobic bacteria indicates the potential for killing anaerobic Mtb. However, how MTZ is activated in Mtb still remains unknown. We aimed to characterize the endogenous nitroreductase responsible for MTZ activation in anaerobic Mtb.MethodsThe minimal inhibitory concentrations (MICs) of Mtb isolates against MTZ were determined by microplate Alamar Blue assay. Intracellular anti-TB activities of MTZ and pyrazinamide (PZA) were tested in THP-1 cells infected by Mycobacterium tuberculosis (Mtb) H37Rv with a multiplicity of infection (MOI) of 10. The nitroreductase activity of purified wild-type Rv3131 and mutants were measured under anaerobic conditions generated by glucose oxidase/catalase system. Two-tailed unpaired Student’s t test was used to compare the difference between various groups.Results180 Mtb isolates (81.8%, 180/220) had MIC values higher than 16 μg/mL, and 40 had MIC values of 16 μg/mL, demonstrating high-level resistance to MTZ under aerobic condition. The number of viable bacteria in macrophages treated with MTZ was dramatically decreased by 71.3% after 5-day MTZ treatment, indicating significant inhibition of MTZ against anaerobic Mtb. In vitro biochemical analysis demonstrated that Rv3131 exhibited the NADPH oxidase activity under anaerobic condition. The substitutions of Cys75Ser and Cys279Ser could maintain 41.7% and 71.1% of enzyme activity compared to wild-type protein, respectively.ConclusionsOur data demonstrate that MTZ has more potent efficacy against intracellular Mtb than PZA. Rv3131 is identified as a nitroreductase enzyme in the activation of MTZ, and Cys75 of Rv3131 is the major active residue for nitroreductase activity.

scholarly journals Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071

1998 ◽  
Vol 42 (12) ◽  
pp. 3234-3241 ◽  
Author(s):  
Chun Y. Tai ◽  
Paul A. Escarpe ◽  
Robert W. Sidwell ◽  
Matthew A. Williams ◽  
Willard Lew ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Neuraminidase Inhibitor ◽  
H3n2 Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Human Influenza ◽  
Wild Type ◽  
Viral Virulence ◽  
High Level

ABSTRACT An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d- N -acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.

scholarly journals Essential role of the linear ubiquitin chain assembly complex and TAK1 kinase in A20 mutant Hodgkin lymphoma

2020 ◽  
Vol 117 (46) ◽  
pp. 28980-28991
Author(s):  
Zhihui Song ◽  
Wei Wei ◽  
Wenming Xiao ◽  
Essel D. Al-Saleem ◽  
Reza Nejati ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Strong Support ◽  
Ubiquitin Chain ◽  
Guide Rna ◽  
Barr Virus ◽  
Protein Ubiquitination ◽  
Crispr Screen ◽  
Epstein Barr

More than 70% of Epstein–Barr virus (EBV)-negative Hodgkin lymphoma (HL) cases display inactivation of TNFAIP3 (A20), a ubiquitin-editing protein that regulates nonproteolytic protein ubiquitination, indicating the significance of protein ubiquitination in HL pathogenesis. However, the precise mechanistic roles of A20 and the ubiquitination system remain largely unknown in this disease. Here, we performed high-throughput CRISPR screening using a ubiquitin regulator-focused single-guide RNA library in HL lines carrying either wild-type or mutant A20. Our CRISPR screening highlights the essential oncogenic role of the linear ubiquitin chain assembly complex (LUBAC) in HL lines, which overlaps with A20 inactivation status. Mechanistically, LUBAC promotes IKK/NF-κB activity and NEMO linear ubiquitination in A20 mutant HL cells, which is required for prosurvival genes and immunosuppressive molecule expression. As a tumor suppressor, A20 directly inhibits IKK activation and HL cell survival via its C-terminal linear-ubiquitin binding ZF7. Clinically, LUBAC activity is consistently elevated in most primary HL cases, and this is correlated with high NF-κB activity and low A20 expression. To further understand the complete mechanism of NF-κB activation in A20 mutant HL, we performed a specifically designed CD83-based NF-κB CRISPR screen which led us to identify TAK1 kinase as a major mediator for NF-κB activation in cells dependent on LUBAC, where the LUBAC-A20 axis regulates TAK1 and IKK complex formation. Finally, TAK1 inhibitor Takinib shows promising activity against HL in vitro and in a xenograft mouse model. Altogether, these findings provide strong support that targeting LUBAC or TAK1 could be attractive therapeutic strategies in A20 mutant HL.

scholarly journals Interdependent Recruitment of SAGA and Srb Mediator by Transcriptional Activator Gcn4p

2005 ◽  
Vol 25 (9) ◽  
pp. 3461-3474 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Fan Zhang ◽  
Gwo Jiunn Hwang ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Wild Type ◽  
General Transcription Factors ◽  
Tata Element ◽  
High Level ◽  
Target Promoters ◽  
Activation Sequence

ABSTRACT Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

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