HUMAN-HERPESVIRUS-8 ORF K8.1 GENE ENCODES IMMUNOGENIC GLYCOPROTEINS GENERATED BY SPLICED TRANSCRIPTS

A cDNA library from phorbol ester-induced human herpesvirus-8 (HHV-8) carrying BCBL-1 cells was screened with an HIV+KS+ serum, and several cDNA clones encoding HHV-8 proteins were identified. Sequence analysis of two full-length cDNA clones show open reading frames (ORFs) encode d by spliced messages originating from the HHV-8 K8.1 gene. One cDNA e ncodes an ORF of 228 amino acids, designated K8.1.A, with a cleavable signal sequence, a transmembrane domain, and four N-glycosylation site s. The splicing event generated the transmembrane domain in the ORF no t seen in the genomic K8.1 ORF. Another cDNA encodes an ORF of 167 ami no acids, designated K8.1.B, that shares similar amino and carboxyl te rmini with ORF K8.1.A but with an in-frame deletion, The primary trans lation products of ORF K8.1A (34 kDa) and K8.1B (20 kDa) in the in vit ro-transcription-translation experiments shifted into glycosylated spe cies of 43 and 32 kDa, respectively, in the presence of microsomal mem branes. This suggested that the ORF K8.1 A and K8.1B encode for glycop roteins. Riboprobes from the K8.1A cDNA insert hybridized with an HHV- 8-specific 0.9-kb abundant transcript from BCBL-1 cells. Synthesis of this RNA was eliminated in the presence of a DNA synthesis inhibitor, suggesting that this RNA was a late gene transcript. Because ORFs K8.1 A and K8.1B are unique for HHV-8, human sera were tested in Western bl ot reactions for antibodies against glutathione-S-transferase-ORF K8.1 A fusion protein. All sera that were positive for HHV-8 antibodies in immunofluorescence assays with phorbol ester-induced BCBL-1 cells were also positive for anti-ORF K8.1A antibodies. This suggests that measu rement of anti-ORF K8.1A antibodies would provide an HHV-8-specific se rological assay. Further work is needed to define the biological role of the HHV-8 ORF K8.1A and K8.1B glycoproteins. (C) 1998 Academic Pres s.