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Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin

S K Dhar1, K Yoshida, Y Machida

  • 1Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Cell
|August 18, 2001
PubMed

Insights

A mutation in the ORC2 gene reduced Orc2 protein, prolonging cell cycle but not affecting cellular DNA replication origins. Viral replication from Epstein-Barr virus origins (oriP) was impaired, showing ORC

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Virology

Background:

  • The Origin Recognition Complex (ORC) is crucial for DNA replication initiation in eukaryotes.
  • Understanding ORC's role in viral DNA replication is essential for comprehending viral lifecycles and developing antiviral strategies.

Purpose of the Study:

  • To investigate the role of the ORC2 gene and its protein product in DNA replication, specifically examining its necessity for viral DNA replication from Epstein-Barr virus (EBV) origins.
  • To determine if the human replication initiation apparatus is involved in the replication of viral origins.

Main Methods:

  • Homologous recombination was used to create a hypomorphic mutation in the ORC2 gene in a human cancer cell line, reducing Orc2 protein levels.
  • Cell cycle progression was analyzed, and the utilization of cellular origins of replication (c-Myc, beta-globin) was assessed.
  • Plasmid replication assays using a construct with the EBV oriP replicator were performed in wild-type and mutant cells.
  • The effect of reintroducing Orc2 protein and the impact of geminin, a replication inhibitor, on oriP replication were evaluated.

Main Results:

  • A hypomorphic mutation in ORC2 significantly decreased Orc2 protein levels (by 90%) and prolonged the G1 phase of the cell cycle.
  • The mutation did not affect the utilization of cellular origins of replication like c-Myc or beta-globin.
  • Cells with reduced Orc2 failed to replicate plasmids containing the EBV oriP, a defect rescued by Orc2 reintroduction.
  • Orc2 was found to associate with oriP, likely via EBNA1, and geminin inhibited oriP replication.

Conclusions:

  • The ORC2 gene product (Orc2) is essential for the replication of the Epstein-Barr virus origin (oriP).
  • The human replication initiation machinery, including ORC, is required for the replication of this viral origin.
  • Orc2's interaction with EBNA1 is likely critical for its role in oriP replication.

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