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

Abstract

Monoclonal 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 vitro transcription 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.

Intracellular Tat of Human Immunodeficiency Virus Type 1 Activates Lytic Cycle Replication of Kaposi's Sarcoma-Associated Herpesvirus: Role of JAK/STAT Signaling

“…The panel of MAbs used included KSHV ORF K8.1 A/B and KSHV ORF59. Both K8.1 and ORF59 MAbs recognize KSHV lytic cycle proteins and have been previously described (10,11). To calculate the percentage of positive cells, photographs of at least 10 unique fields were taken of every slide, and the number of positive and negative cells was counted separately by three individuals, including one who was blinded to the results.…”

Molecular Genetics of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Epidemiology and Pathogenesis

“…Indeed, most infected cells in KS specimens display latent KSHV gene expression, with occasional cells expressing lytic transcripts (86,143,249,301,384,389,483,489), suggesting that the low level of spontaneous lytic gene expression is not an artifact of tissue culture models. More recent models of de novo infection of cultured endothelial cells have also demonstrated a similar mixed pattern of gene expression (106,278,358).…”

Envelope Glycoprotein gB of Kaposi's Sarcoma-Associated Herpesvirus Is Essential for Egress from Infected Cells

“…Rabbit polyclonal antibodies against KSHV ORF 73 (LANA-1) (2, 36, 57) and full-length gB proteins (58) and monoclonal antibodies against KSHV-gpK8.1A and ORF 59 (11,63) proteins were used in this study.…”