Activation of human herpesvirus 8 open reading frame K5 independent of ORF50 expression

Open reading frame (ORF) 50 of human herpesvirus 8 (HHV8, Kaposi's sarcoma-associated herpesvirus) is one of the immediate-early gene and a homologue of BRLF1 gene of Epstein-Barr virus. It encodes a key switch protein to trigger viral lytic replication from latency. We have established several hybridoma clones producing monoclonal antibodies (MAbs) to the products of HHV8 ORFs. Using these antibodies, we analyzed antigen expression in a HHV8 infected cell line after treatment with phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA). A MAb reacted to 110 kilodalton (kDa) and 62 kDa proteins encoded by ORF50 (ORF50 protein). Kinetic studies of antigen expression by Western blotting revealed that ORF50 protein was induced as early as 6 h after TPA treatment. The proteins encoded by ORFK3, ORFK5, ORFK9, ORF59 and ORFK8.1 were not detected earlier than ORF50 protein. However, when antigen positive cells were counted by immunofluorescent antibody (IFA) test, number of ORFK5 protein positive cells were higher than that of ORF50 protein positive cells at all time after TPA or mock treatment. To confirm the results of IFA test, individual cell was analyzed by reverse transcription polymerase chain reaction. Some cells expressed ORFK5 transcript but not ORF50 transcript. Therefore, we concluded that, although ORF50 protein is a key switch protein of ORFK3, ORFK9, ORF59 and ORFK8.1 expression, it is not essential to trigger ORFK5 gene.