title: Human herpesvirus 8 glycoprotein K8.1: expression, post-translational modification and localization analyzed by monoclonal antibody.
abstract: BACKGROUND
  The genome of human herpesvirus 8 (HHV-8) contains at least 84 open reading frames, including the highly immunogenic K8.1. Other studies have 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, post-translational modification and localization of HHV-8 gpK8.1 by monoclonal antibody (mAb).
  
  
  METHODS
  Mabs to HHV-8 produced by conventional hybridization and several clones identified. 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 identified a 0.75-kb insert from the lambdaZAP cDNA expression library of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced BCBL-1 cells. Sequence analysis revealed that the cDNA insert corresponds to the published spliced ORF K8.1 mRNA of HHV-8. By immunofluorescence assay, the mAb stained the cell membrane, cytoplasm and perinuclear region of TPA induced BCBL-1 cells and showed 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 rMW 35/37 kDa (gpK8.1B). Both species were labeled by [14C]glucosamine, indicating that they are glycosylated and only gpK8.1A was detected in the virions. Expression of the full length K8.1 derived from cDNA in baculovirus system confirmed that these two glycoproteins are encoded by K8.1 gene. Enzymatic deglycosylation with endoF/peptide-N-glycosidase F, led to the reduction of rMW of both polypeptides whereas deglycosylation with O-glycosidase led only the reduction of rMW of K8.1A.
  
  
  CONCLUSIONS
  The mAb 19B4 reacts specifically with BCBL-1 and Sf9 cells infected with recombinant baculovirus containing HHV-8 K8.1 gene. In several assays the mAb reacts with gpK8.1A and gpK8.1B. Only the mature spliced gpK8.1A is incorporated into virion. Enzymatic deglacosylation determined that gpK8.1A is N- and O-glycosylated, whereas gpK8.1B may lack O-glycosylation.
authors.affiliation: Viral and Rickettsial Disease Laboratory Branch, Division of Communicable Disease Control, California State Department of Health Services, Berkeley 94704, USA.
authors.forename: L
authors.initials: L
authors.lastname: Wu
authors.forename: R
authors.initials: R
authors.lastname: Renne
authors.forename: D
authors.initials: D
authors.lastname: Ganem
authors.forename: B
authors.initials: B
authors.lastname: Forghani
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology*; D002467:Cell Nucleus / Q000821:virology*; D003593:Cytoplasm / Q000821:virology*; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D019288:Herpesvirus 8, Human / Q000187:drug effects / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D016366:Open Reading Frames; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000032:analysis; D018411:Spodoptera / Q000821:virology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014771:Virion / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D006023:Glycoproteins; C113454:K8.1 protein, Human herpesvirus 8; D020033:Protein Isoforms; D012367:RNA, Viral; D014764:Viral Proteins; D006026:Glycoside Hydrolases; D013755:Tetradecanoylphorbol Acetate