Human herpesvirus 8 glycoprotein K8.1: expression, post-translational modification and localization analyzed by monoclonal antibody

Background: The genome of human herpesvirus 8 (HHV-8) contains at least 84 open reading frames, including the highly immunogenic K8.1. Other studies h ave determined that K8.1 gene generates at least two spliced transcripts in the HHV-8 infected BCBL-1 cells, termed as glycoprotein (gp)K8.1A and gpK8 .1B. Objective: To analyze the expression, past-translational modification and localization of HHV-8 gpK8.1 by monoclonal antibody (mAb). Study design : Mabs to HHV-8 produced by conventional hybridization and several clones i dentified. A mAb was used by various immunological assays to analyze HHV-8 K8.1 proteins in BCBL-1- and Sf9 insect cells. Results: MAb clone 19B4 iden tified a 0.75-kb insert from the lambda ZAP cDNA expression library of 12-O -tetradecanoylphorbol-13-acetate (TPA)-induced BCBL-1 cells. Sequence analy sis revealed that the cDNA insert corresponds to the published spliced ORF K8.l mRNA of HHV-8. By immunofluorescence assay, the mAb stained the cell m embrane, cytoplasm and perinuclear region of TPA induced BCBL-1 cells and s howed no cross-reactivity with other herpesviruses. By immunoblotting assay mAb 19B4 reacted with two species polypeptides giving a diffuse band with rMW from 42 to 64 kDa (gpK8.1A) and two closely migrating polypeptides of r MW 35/37 kDa (gpK8.1B). Both species were labeled by [C-14]glucosamine, ind icating that they are glycosylated and only gpK8.1A was detected in the vir ions. Expression of the full length K8.1 derived from cDNA in baculovirus s ystem confirmed that these two glycoproteins are encoded by K8.1 gene. Enzy matic deglycosylation with endoF/peptide-N-glycosidase F, led to the reduct ion of rMW of both polypeptides whereas deglycosylation with O-glycosidase led only the reduction of rMW of K8.1A. Conclusion: The mAb 19B4 reacts spe cifically with BCBL-1 and Sf9 cells infected with recombinant baculovirus c ontaining HHV-8 K8.1 gene. In several assays the mAb reacts with gpK8.1A an d gpK8.1B. Only the mature spliced gpK8.1A is incorporated into virion. Enz ymatic deglacosylation determined that gpK8.1A is N- and O-glycosylated, wh ereas gpK8.1B may lack O-glycosylation. (C) 2000 Elsevier Science B.V. All rights reserved.