Identification and expression of the human herpesvirus 6 glycoprotein H and interaction with an accessory 40K glycoprotein.
In herpes simplex virus (HSV) the small secreted glycoprotein gL forms a heterodimer with the transmembrane envelope glycoprotein gH. Here we identify the human herpesvirus 6 (HHV-6) gL gene, express HHV-6 gL and gH homologues, and examine interactions between HHV-6 gH and gL. The HHV-6 gL gene encoded a glycoprotein with an amino acid sequence which showed closest similarity to the human cytomegalovirus (HCMV) gL homologue (18% identity). Products of HHV-6 gH and gL genes were characterized in an in vitro transcription-translation system and in a transient in vivo expression system. Both gH and gL were transcribed and translated in vitro to give products of apparent M(r) of 65K and 28K in SDS-PAGE, and these could be processed by addition of microsomes to 110K and 40K, respectively. To study gH/gL interactions, gH was tagged with the nine amino acid epitope for monoclonal antibody LP14 (anti-HSV-1 gD). LP14 and a human serum sample specifically immunoprecipitated gH and a stable complex of gH and gL co-expressed in an in vivo vaccinia virus-T7 system. The gH and gL produced in this in vivo expression system corresponded to the M(r)s of the fully processed glycoproteins identified in the in vitro system. The gH expressed together with gL was recognized by human sera more easily than when examined on its own in immunofluorescence assays. Dual expression of gH and gL in transfected T lymphocytes (JJhan) caused reactions with 75% of human sera tested (12 HHV-6-positive, HCMV-negative serum samples), but gL expressed alone was not recognized by these sera. The immunofluorescence studies also showed that the glycoproteins were localized in Golgi-like bodies in fibroblasts, but occurred throughout the endoplasmic reticulum in T lymphocytes, the normal cellular target for HHV-6. These results show the identification of the HHV-6 homologue to the HCMV and HSV gL genes, identification and production of HHV-6 gH and gL expressed both in vitro and in vivo, complex formation between these glycoproteins, and evidence that this complex may be localized differently in fibroblasts as compared to T lymphocytes and that it is immunogenic.
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, U.K.
D X
DX
Liu
U A
UA
Gompels
J
J
Nicholas
C
C
Lelliott
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016026:Bone Marrow Transplantation; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D015654:Herpesvirus 6, Human / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C070917:glycoprotein H, herpesvirus 6