Use of antigen expressed in bacteria for detection of EBV‐specific thymidine kinase antibodies in sera from patients with nasopharyngeal carcinoma

Hsu, Ting-Yu; Pai, Chien-Ying; Shieh, Shing-Mey; Cho, Show-Mei; Chen, Jen‐Yang; Yang, Czau‐Siung

doi:10.1002/jmv.1890380311

Cited by 11 publications

(11 citation statements)

References 36 publications

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“…The TK activity assay was performed as described previously [38]. Proteins of TK and mutants, which were expressed by pRsetA vector, had the histidine-tagged polypeptides at the N-terminus.…”

Section: Tk Activity Assaymentioning

confidence: 99%

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Chen

Chang

et al. 2004

Biochemical Journal

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Thymidine kinase (TK), encoded by EBV (Epstein-Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp 392 → Glu) and R393H retain activity, indicating that a negative charge is important for Asp 392 and a positive charge is required for Arg 393 . The increased binding affinities of these two mutants for 3 -deoxy-2 ,3 -didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp 392 plays multiple roles in this region. His 394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60 % relative activity. Strikingly, when Phe 402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3 -azido-3 -deoxythymidine, iododeoxyuridine and β-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe 402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

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Section: Tk Activity Assaymentioning

confidence: 99%

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Chen

Chang

et al. 2004

Biochemical Journal

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“…The cloned EBV TK gene, which was derived from P3HR1 cells, was a gift from J. Y. Chen, College of Medicine, National Taiwan University (22,33). Approximately 100 ng of PET-TK B1B plasmid was used to transform competent cells, the BL21(DE3)/pLysS expression host, by the standard procedure of Sambrook et al (42).…”

Section: Epstein-barr Virus (Ebv) Is An Important Human Pathogenmentioning

confidence: 99%

Anti-Epstein-Barr Virus (EBV) Activity of β- l -5-Iododioxolane Uracil Is Dependent on EBV Thymidine Kinase

Kira

Grill

Dutschman

et al. 2000

Antimicrob Agents Chemother

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␤-L-5-Iododioxolane uracil was shown to have potent anti-Epstein-Barr virus (EBV) activity (50% effective concentration ‫؍‬ 0.03 M) with low cytotoxicity (50% cytotoxic concentration ‫؍‬ 1,000 M). It exerts its antiviral activity by suppressing replicative EBV DNA and viral protein synthesis. This compound is phosphorylated in cells where the EBV is replicating but not in cells where the EBV is latent. EBV-specific thymidine kinase could phosphorylate ␤-L-5-iododioxolane uracil to the monophosphate metabolite. The K m of ␤-L-5-iododioxolane uracil with EBV thymidine kinase was estimated to be 5.5 M, which is similar to that obtained with thymidine but about fivefold higher than that obtained with 2 fluoro-5-methyl-␤-L-arabinofuranosyl uracil, the first L-nucleoside analogue discovered to have anti-EBV activity. The relative V max is seven times higher than that of thymidine. The anti-EBV activity of ␤-L-5-iododioxolane uracil and its intracellular phosphorylation could be inhibited by 5-ethynylthymidine, a potent EBV thymidine kinase inhibitor. The present study suggests that ␤-L-5-iododioxolane uracil exerts its action after phosphorylation; therefore, EBV thymidine kinase is critical for the antiviral action of this drug. Epstein-Barr virus (EBV) is an important human pathogen.This virus has been determined to cause infectious mononucleosis, fatal acute infectious mononucleosis-X-linked lymphoproliferative syndrome, and oral hairy leukoplakia (16,17,48). EBV also has a close association with several types of malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), Hodgkin's disease, (2, 13, 21), some T-cell lymphomas (1,26,32,35,40,58), smooth muscle cell leiomyosarcoma (23), and certain cases of gastric adenocarcinomas (38,46,53). EBV infection can enhance human immunodeficiency virus type 1 (HIV-1) replication in T cells (56), and EBV is also related to the development of lymphoma induced in AIDS patients (55). It was also reported that almost all posttransplant lymphomas appeared to be EBV genome positive, irrespective of histological appearance or clonability of the lesion (18,44,49,59). A more complex picture of EBV-associated malignancies is emerging, particularly with regard to virus-positive tumors of non-B-cell origin. It is hoped that a better understanding of EBV persistence and the part played by EBV in the oncogenic process will permit the development of new approaches aimed at the prevention and treatment of EBV-associated tumors. Therefore, it would be ultimately useful to have anti-EBV compounds that do not have serious side effects.Several compounds have shown anti-EBV activity in cell culture systems, including acyclovir (ACV), ganciclovir (DHPG), 2Ј-fluoro-5-methyl-␤-D-arabinofuranosyl uracil (D-FMAU), and (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (cidofivir) (6,9,26,27,36,50,52). However, the clinical application of many of these compounds is restricted by severe side effects (52). Recently, our laboratory found a new anti-EBV L-dioxolane nucleoside analogue, ␤...

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“…These were based on antibodies to different antigenic components of the EBV (9,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Attempts to use panels of antibody-based markers have generally been useful in improving the accuracy of NPC detection (13, 15, 20, 21, 29 -32 ).…”

mentioning

confidence: 99%

Improved Accuracy of Detection of Nasopharyngeal Carcinoma by Combined Application of Circulating Epstein–Barr Virus DNA and Anti-Epstein–Barr Viral Capsid Antigen IgA Antibody

Leung¹,

Tam²,

Chan³

et al. 2004

Clinical Chemistry

101

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Use of antigen expressed in bacteria for detection of EBV‐specific thymidine kinase antibodies in sera from patients with nasopharyngeal carcinoma

Cited by 11 publications

References 36 publications

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Anti-Epstein-Barr Virus (EBV) Activity of β- l -5-Iododioxolane Uracil Is Dependent on EBV Thymidine Kinase

Improved Accuracy of Detection of Nasopharyngeal Carcinoma by Combined Application of Circulating Epstein–Barr Virus DNA and Anti-Epstein–Barr Viral Capsid Antigen IgA Antibody

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