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Expression of COS-1 cells of Epstein-Barr virus nuclear antigen from a complete gene and a deleted gene
Journal article   Open access   Peer reviewed

Expression of COS-1 cells of Epstein-Barr virus nuclear antigen from a complete gene and a deleted gene

M. F Robert, D Shedd, R. J Weigel, D. K Fischer and G Miller
Journal of virology, Vol.50(3), pp.822-831
1984
DOI: 10.1128/jvi.50.3.822-831.1984
PMCID: PMC255742
PMID: 6328012
url
https://europepmc.org/articles/pmc255742View
Published (Version of record) Open Access

Abstract

In a previous study the BamHI-K fragment of Epstein-Barr virus DNA was shown to induce a nuclear antigen, Epstein-Barr virus nuclear antigen (EBNA), when cotransfected with the herpes simplex virus thymidine kinase gene into mouse LTK- cells. We have now inserted the BamHI-K fragment and a BamHI/HindIII subfragment, I1f, into shuttle vectors containing the origin of replication of simian virus 40. These plasmids have been introduced into COS-1, which are monkey kidney cells transformed by an origin-defective simian virus 40 genome. This expression system permitted rapid characterization of antigens, mRNAs, and proteins related to EBNA. The same-sized EBNA protein (~78,000) was made after transfection with BamHI-K (5.2 kilobase pairs [kbp]) or the I1f subfragment (2.9 kbp). A deletion of about 600 bp occurred with the I1f fragment was propagated on the pSV2 plasmid in Escherichia coli. The deleted fragment gave rise to a smaller protein (~52,000). These data provide evidence that EBNA is encoded by the 2.9-kbp I1f and is not an induced cellular protein. Nuclear antigen and polypeptide expression occurred equally well when the Epstein-Barr virus DNA was cloned on PSV2-gpt or pSVOd. The latter plasmid lacks sequences allowing for efficient early gene transcription as well as splicing and polyadenylation signals which are present in pSV2. Preliminary mapping of the EBNA gene transcripts demonstrated that two mRNAs (2.9 and 2.4 kilobases [kb]) are homologous to the I1f fragment. Taken together, the data suggest that the 2.9-kbp I1f fragment contains the structural for EBNA synthesis. COS-1 cells will thus provide a valuable system in which to analyze functional domains of the EBNA gene.
 Genetics Microbiology Virology Biological and medical sciences Fundamental and applied biological sciences. Psychology

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