Identification and characterization of a cDNA derived from multiple splicing that encodes envelope glycoprotein gp105 of human herpesvirus 6.
The glycoprotein complex gp82-gp105 is a major virion envelope glycoprotein complex of human herpesvirus 6 variant A (HHV-6A) and consists of a number of related polypeptides. Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against this glycoprotein complex neutralized HHV-6A infectivity. We have previously reported the isolation, mapping, and characterization of a portion of the viral genomic DNA fragment encoding the gp82-gp105 complex and the identification of the neutralizing epitope (B. Pfeiffer, Z. N. Berneman, F. Neipel, C. K. Chang, S. Tirwatnapong, and B. Chandran, J. Virol. 67:4611-4620, 1993). This gene was further characterized by the identification of a 2.3-kb genomic fragment and by the identification of a 2.5-kb cDNA clone. The genomic sequence contains a short open reading frame (ORF) encoding the epitope recognized by the MAbs. The identified cDNA showed specificity for HHV-6 in Southern blot analysis with viral DNA. In Northern (RNA) blot analysis with total RNA from HHV-6A(GS)-infected cells, the cDNA insert specifically hybridized with several RNA species. Restriction mapping analysis localized this cDNA to the HHV-6A(U1102) genomic BamHI G fragment, at the right end of the unique long segment of the genome and to the SalI L and SalI O fragments within the left and right terminal direct repeat regions, respectively. In vitro transcription and translation of the cDNA revealed a polypeptide of about 88.5 kDa which was glycosylated in the presence of microsomal membranes to a polypeptide of approximately 104.2 kDa. Both polypeptides were immunoprecipiated by MAb 2D6, verifying the identity of the cDNA as encoding the gp105 in the gp82-gp105 complex. Sequence analysis of the cDNA revealed a large ORF potentially encoding a 650-amino-acid protein with 11 potential N-linked glycosylation sites and 18 cysteine residues. A potential membrane-spanning domain is located only near the amino terminus of the putative protein, indicating that gp105 may be a class 2 glycoprotein. Comparison of the cDNA nucleotide sequence with sequences from HHV-6A(U1102) genomic BamHI G and SalI L fragments revealed that the gene encoding gp105 contains 12 exons, spanning over 20 kb of the viral genome, with intron 1 spanning about 8 kb of genomic DNA. The first exon of the cDNA mapped to the right and left terminal direct repeats, while the other exons mapped within the unique long segment of the genome.(ABSTRACT TRUNCATED AT 400 WORDS)
Pfeiffer
B
B
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160, USA.
Thomson
B
B
Chandran
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004279:DNA, Viral; D015654:Herpesvirus 6, Human / Q000235:genetics*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics*
D018076:DNA, Complementary; D004279:DNA, Viral; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D014759:Viral Envelope Proteins; C093520:glycoprotein 105, Human herpesvirus 6