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Immunological characterization of the Epstein-Barr virus phosphoprotein PP58 and deoxyribonuclease expressed in the
Abstract:
The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells. The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E. coli. DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities. Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58. The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E. coli. The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, B95-8, M-ABA and BL74 induced by TPA and n-butyrate. The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0). Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera. Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC. In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.
Insights
Epstein-Barr virus (EBV) proteins pp58 and BGLF5 were expressed in insect cells. Antibodies against pp58 and EBV DNase were detected in nasopharyngeal carcinoma (NPC) patients, suggesting diagnostic potential.
Area of Science:
- Molecular biology
- Virology
- Immunology
Background:
- Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC).
- Understanding EBV protein functions and their role in NPC pathogenesis is crucial.
Purpose of the Study:
- To clone, express, and characterize EBV phosphoprotein pp58 (BMRFI) and deoxyribonuclease (BGLF5).
- To investigate the potential of these proteins as diagnostic markers for NPC.
Main Methods:
- Cloning of EBV open reading frames into baculovirus vectors.
- Expression in Spodoptera frugiperda insect cells.
- Characterization using DNA-cellulose chromatography, immunofluorescence, immunoblot, ELISA, and enzyme activity assays.
Main Results:
- Recombinant pp58 exhibited DNA-binding activity and was recognized by NPC patient sera.
- Recombinant EBV DNase showed exonuclease and endonuclease activities, dependent on magnesium and optimal at pH 8.0.
- NPC patient sera inhibited EBV DNase activity, indicating potential for diagnostic assays.
Conclusions:
- Recombinant EBV proteins pp58 and BGLF5 can be expressed and characterized.
- Antibodies against pp58 and EBV DNase are present in NPC patients.
- Recombinant EBV DNase shows promise for NPC detection through enzyme activity inhibition assays.