| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 93 === Epstein-Barr virus (EBV) BALF3 is a homologue of the herpes simplex virus (HSV) UL28 gene, which encodes a putative terminase component conserved among herpesviruses. Terminase plays a role in recognition of pac sequences within the DNA termini of herpevirus, and in cleavage of the concatemeric DNA. The cleavage is required for correct package of viral genome and maturation of viral nucleocapsid. However, the expression of BALF3 has not been demonstrated previously. An anti-sense RNA probe detected a specific 6.0 kb transcript in Akata (EBV+) 6 h post-induction. A transcript containing BALF3 was also detected by RT-PCR using specific RT primers. These results suggest BALF3 containing transcript is expressed in EBV replicating cells, and the transcript is probably driven by BALF2 promoter. To characterize the expression of BGLF3 protein in EBV replicating cells, we then generated BALF3 specific sera. His-tag fused BALF3 protein was expressed in Escherichia coli and purified with nickel beads. The purified protein was used to generate mouse and rabbit specific antisera. Using BALF3 specific sera, we detected the expression of Flag-BALF3 fusion protein in transfectly transfected-293T cells in immunoblotting; and the fusion protein was observed in the cytoplasm of transfected cells in immunoflourescence assay. BALF3 was also detected with specific serum in EBV positive Akata and NA cells, and the detected molecular weight of BALF3 is about 75-kDa. Simultaneous detection of expression kinetics of BALF3 and other viral proteins revealed that BALF3 expresses as an early protein. Finally, we tested whether BALF3 antibody is present in the sera of nasopharyngeal carcinoma (NPC) patients using bacterially purified recombinant BALF3 protein as antigen. As a result anti-BALF3 IgA was detected in 4 of 16 (25%) and IgG was detected in 10 of 16 (62%) patients, but not detected in all 9 healthy donors. This observation further supports the in vivo expression of BALF3 in EBV infected individuals. Further studies are required to identify the sub-cellular localization and biological functions of BALF3 within EBV replicating cells.
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