Cloning and expression of a cDNA encoding the Epstein-Barr virus thymidine kinase gene

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

doi:10.1016/0166-0934(92)90012-3

Cited by 9 publications

(7 citation statements)

References 23 publications

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“…As expected, the BamHl X assay was positive, and our results thus validated those of Liu et al [6] and of Tung and Summers [7] who used the slightly shorter LORF. The activity we observed with SORF, however, clearly shows that the 243 amino-terminal residues of the 607-residue LORF product are not required for dTK activity.…”

Section: S O R F a C A T T A A A T C A T Q Q T T G G Q Q A T C Q Q A supporting

confidence: 79%

“…Balasubramaniam et al [5] compared the predicted amino acid sequences of 12 herpesviral dTKs and identified 6 conserved regions com mon to all 12, including those of EBV and the related Her pesvirus saimiri (HVS). Two additional reports of dTK activity in dTK-E. coli transfected with the EBV BXLF1 ORF have since appeared [6,7], with a size of 67-70 kD, A preliminary report of this work was given at the July 1995 meeting of the American Society for Virology in Austin, Tex., USA. [3.…”

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confidence: 99%

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The Epstein Barr Virus Genome Encodes Deoxythymidine Kinase Activity in a Nested Internal Open Reading Frame

Holton¹,

Gentry²

1996

Intervirology

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The Epstein-Barr virus (EBV) BXLF1 fragment open reading frame (LORF), thought to encode deoxythymidine kinase (dTK) activity, and a shorter frame (SORF), starting at an internal in-frame AUG, were isolated by polymerase chain reaction from a plasmid containing the EcoR1 fragment of EBV strain FF-41. These were transfected into dTK^–Escherichia coli, producing multiple SORF- or LORF-containing colonies, which expressed dTK. The 243 NH₂ terminal residues of the LORF-encoded polypeptide thus are not essential for dTK activity. SORF, with 1,092 bp, is predicted to encode a 36- to 37-kD polypeptide, in the size range of other herpesviral dTKs.

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Section: S O R F a C A T T A A A T C A T Q Q T T G G Q Q A T C Q Q A supporting

confidence: 79%

mentioning

confidence: 99%

The Epstein Barr Virus Genome Encodes Deoxythymidine Kinase Activity in a Nested Internal Open Reading Frame

Holton¹,

Gentry²

1996

Intervirology

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“…SF9 cells derived from Spodoptera frugiperda were propagated in TC-100 medium (Gibco BRL, Grand Island, NY) containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Complementary DNA (cDNA) fragments, encoding the EBV EA-D complex (DNA sequence coordinates 79,819-81,110 of B95-8 strain), DNase (120,410),or TK (142,861) were inserted into plasmid pVL1393, a baculovirus transfer vector [Liu et al, 1992;Lin et al, 1995]. The resulting recombinant plasmids were co-transfected with the wild-type baculovirus genome into SF9 cells as instructed by the manufacturer of Baculo Gold transfection kits (Pharmingen, San Diego, CA).…”

Section: Expression Of Ebv Lytic Proteins In Baculovirus Systemmentioning

confidence: 99%

Immune responses to Epstein–Barr virus lytic proteins in patients with nasopharyngeal carcinoma

Huang

Sheen

Chen

et al. 2004

Journal of Medical Virology

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Immune responses to three Epstein-Barr virus (EBV) lytic proteins, DNase, thymidine kinase (TK), and BMRF-1 gene products (50/52 kDa diffused early antigen, EA-D complex) were determined in EBV-infected control individuals and patients with nasopharyngeal carcinoma (NPC). Immunofluorescence assays (IFA) were used to detect their humoral immune responses using recombinant EBV lytic proteins expressed in a baculovirus system as antigens. Cell proliferation assays were performed to evaluate their cellular immune responses by monitoring 3H-thymidine incorporation. Seventy patients with NPC and 32 non-cancer controls were analyzed. The results of IFA showed antibody titers to all three EBV lytic proteins to be higher in the patients with NPC especially for the IgA class. Positivity rates of the three IgA antibodies also were higher in the patients with NPC population. Furthermore, the profiles of the IgA antibodies correlated with those to total early antigens (EA) expressed in the early phase and viral capsid antigen (VCA) expressed in the late phase, of EBV replication. The most interesting finding was that antibody titers to the three EBV lytic proteins were associated significantly with metastases of cervical lymph nodes in patients with NPC. As for cellular immunity to the EA-D complex and DNase, weak responses were observed in the cell proliferation assays. Peripheral blood cells from most individuals could not be stimulated to proliferate, except for a few patients with NPC whose antibody titers against the EA-D complex and DNase also were very high.

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“…The template for PCR was the plasmid pET-TKB1B, which encodes a full-length, 607-amino-acid TK protein [37]. A mutated complementary set of primers was designed for each specific wild-type residue (Table 1).…”

Section: Site-directed Mutagenesismentioning

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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Cloning and expression of a cDNA encoding the Epstein-Barr virus thymidine kinase gene

Cited by 9 publications

References 23 publications

The Epstein Barr Virus Genome Encodes Deoxythymidine Kinase Activity in a Nested Internal Open Reading Frame

The Epstein Barr Virus Genome Encodes Deoxythymidine Kinase Activity in a Nested Internal Open Reading Frame

Immune responses to Epstein–Barr virus lytic proteins in patients with nasopharyngeal carcinoma

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

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