Mitosis-specific hyperphosphorylation of Epstein-Barr virus nuclear antigen 2 suppresses its function

Area of Science:

• Virology
• Cell Biology
• Molecular Biology

Background:

• Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is crucial for EBV type III latency and B lymphocyte immortalization.
• EBNA-2 transactivates viral and cellular gene promoters, driving cell proliferation.

Purpose of the Study:

• To investigate the cell-cycle regulation of EBNA-2.
• To determine the role of p34(cdc2) kinase in EBNA-2 phosphorylation.
• To assess the functional consequences of EBNA-2 hyperphosphorylation on gene transactivation and protein interactions.

Main Methods:

• Coimmunoprecipitation assays to detect protein-protein interactions between EBNA-2 and p34(cdc2).
• In vitro kinase assays using purified p34(cdc2)/cyclin B1 to phosphorylate EBNA-2.
• Analysis of EBNA-2 transactivation activity on the LMP-1 promoter.
• Measurement of EBNA-2 association with PU.1.
• Quantification of endogenous LMP-1 mRNA levels in different cell cycle phases.

Main Results:

• EBNA-2 is specifically hyperphosphorylated during mitosis.
• Evidence suggests p34(cdc2) kinase is involved in EBNA-2 hyperphosphorylation through physical association and in vitro phosphorylation.
• Hyperphosphorylation of EBNA-2 reduces its transactivation of the LMP-1 promoter by approximately 50%.
• EBNA-2's association with PU.1 is decreased by about 50% in M-phase-arrested cells.
• Endogenous LMP-1 mRNA levels are reduced in M-phase cells.

Conclusions:

• EBNA-2 undergoes cell-cycle-dependent hyperphosphorylation, likely mediated by p34(cdc2) kinase during mitosis.
• Mitotic hyperphosphorylation of EBNA-2 impairs its transcriptional activity and protein binding, affecting viral gene expression.
• These findings suggest a mechanism for regulating EBV gene expression during the cell cycle in type III latency.