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Definitive identification of a member of the Epstein-Barr virus nuclear protein 3 family
Abstract:
Some Epstein-Barr virus (EBV) immune human antisera are known to react with a 142-kDa protein, EBV-encoded nuclear antigen 3 (EBNA3), which, like EBNA1 and EBNA2, is likely to be involved in the establishment of latent infection or growth transformation. We have now constructed gene fusions between Escherichia coli lacZ and an EBV DNA open reading frame (BERF1; BamHI E fragment rightward open reading frame 1), which is transcribed into an mRNA in latently infected cells. Purified hybrid protein from one of these constructs, chosen because of its reactivity with EBNA3-positive human antisera, was used to affinity purify the specific antibody from human antiserum. This specific antibody was used to prove that EBNA3 is encoded, at least in part, by BERF1, and that EBNA3 is in the nucleus of each latently infected cell. In rodent cells, BERF1 encodes a 120- to 130-kDa protein, which translocates to the nucleus and is recognized by EBNA3-positive human antisera. Two other proteins similar in size to EBNA3 are detected in latently infected cells by EBV immune human antisera. Two EBV open reading frames related to BERF1 may encode these proteins.
Insights
Researchers identified the Epstein-Barr virus (EBV) nuclear antigen 3 (EBNA3) protein, crucial for latent infection, as being encoded by the BERF1 gene. This EBNA3 protein is located in the nucleus of infected cells.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Epstein-Barr virus (EBV) latent infection involves nuclear antigens like EBNA1, EBNA2, and EBNA3.
- Human antisera recognize a 142-kDa EBNA3 protein, suggesting its role in viral latency and cell transformation.
Purpose of the Study:
- To investigate the genetic origin and cellular localization of the EBV-encoded nuclear antigen 3 (EBNA3).
- To confirm the association between the BERF1 open reading frame and the EBNA3 protein.
Main Methods:
- Construction of Escherichia coli lacZ-EBV DNA (BERF1) gene fusions.
- Affinity purification of specific antibodies using hybrid proteins.
- Western blot analysis and immunofluorescence to detect protein expression and localization.
Main Results:
- A specific antibody confirmed that EBNA3 is encoded, at least partially, by the BERF1 open reading frame.
- EBNA3 was localized to the nucleus of latently infected cells.
- BERF1 expressed a 120-130 kDa protein in rodent cells, which localized to the nucleus and was recognized by EBNA3-positive antisera.
Conclusions:
- The BERF1 open reading frame encodes the EBNA3 protein, a key component of EBV latency.
- EBNA3 resides in the nucleus, consistent with its proposed role in regulating viral gene expression and cell transformation.
- Related EBV open reading frames may encode other EBNA3-like proteins recognized by human antisera.