Identification and Characterization of Human Herpesvirus-8 Lytic Cycle-Associated ORF 59 Protein and the Encoding cDNA by Monoclonal Antibody

AbstractMonoclonal antibody (Mab) 11D1 specific for HHV-8 showed a predominantly nuclear membrane fluorescence with about 30% of phorbol ester (TPA)-induced HHV-8-carrying BCBL-1 cells and with 2–8% of uninduced cells, but not with other herpes viruses infected cells. This Mab immunoprecipitated a 50-kDa polypeptide from BCBL-1 cells. The synthesis of this polypeptide was reduced but not inhibited by phosphonoacetic acid (PAA). A 2.3-kb cDNA insert from a cDNA library of TPA-induced BCBL-1 cells was identified by Mab 11D1. Sequence analysis shows that this cDNA is open at the 5′ end and encodes two ORFs of 396AA (5′ end) and 357AA (3′ end). These ORFs are identical to the published HHV-8 ORFs 59 and 58, respectively.In vitrotranscription and translation of the cDNA resulted in the synthesis of a 50-kDa polypeptide and its partial peptide map was identical to that of the 50-kDa polypeptide detected in the TPA induced BCBL-1 cells. Riboprobe made from the cDNA insert hybridized with several viral specific RNAs from BCBL-1 cells. Levels of these RNA species were reduced, but not inhibited by PAA. These characteristics are similar to other herpes viruses genes encoding the lytic cycle associated early–late class accessory proteins that are essential for viral DNA replication. This Mab 11D1 recognizing the HHV-8 lytic cycle associated ORF 59 protein will be highly useful in monitoring the lytic replicative cycle