Cloning and characterization of cDNA clones corresponding to transcripts from the BamHI G region of the Epstein-Barr virus genome and expression of BGLF2.
A cDNA library was constructed from poly(A)+ RNA isolated from the iododeoxyuridine-treated P3HR1 cell line. Five cDNA clones, which hybridized with the BamHI G fragment of Epstein-Barr virus (EBV) DNA, were subcloned and sequenced. Clones G2, G3 and G4 corresponded to the BGLF2 open reading frame (ORF) of EBV (B95-8, nucleotides 126,837 to 125,866); G3 was found to contain the entire BGLF2 ORF. The predicted Mr of the putative protein product of the EBV B95-8 BGLF2 ORF is 36K. Complete nucleotide sequencing of G3 revealed that there were two nucleotide changes from the reported sequence of the EBV B95-8 BGLF2 gene, but these did not alter the predicted amino acid sequence of the products. Clone G3 and a cDNA derived from it by N-terminal deletion were expressed in Escherichia coli, producing fusion proteins. Rabbit antisera against these proteins were shown to react with viral capsid antigen-expressing HR1 cells in an indirect immunofluorescence assay. In vitro transcription/translation products and fusion proteins expressed in E. coli were used to determine the presence of antibodies in sera from EBV-infected individuals. The results of immunoprecipitation and immunoblotting studies showed that the majority of EBV-seropositive individuals mount a serum antibody response to the BGLF2 ORF-encoded protein.
Chen
M R
MR
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Republic of China.
Hsu
T Y
TY
Lin
S W
SW
Chen
J Y
JY
Yang
C S
CS
D000595:Amino Acid Sequence; D001345:Autoradiography; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015245:Deoxyribonuclease BamHI; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014760:Viral Fusion Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
C072324:BGLF2 protein, Epstein-barr virus; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D004247:DNA; D015245:Deoxyribonuclease BamHI