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E2F proteins are posttranslationally modified concomitantly with a reduction in nuclear binding activity in cells
S J Advani1, R R Weichselbaum, B Roizman
1The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Chicago, Illinois 60637, USA.
Abstract:
The transition from G(1) to S phase in the cell cycle requires sequential activation of cyclin-dependent kinase 4 (cdk4) and cdk2, which phosphorylate the retinoblastoma protein, causing the release of E2F. Free E2F upregulates the transcription of genes involved in S phase and cell cycle progression. Recent studies from this and other laboratories have shown that herpes simplex virus 1 stabilizes cyclin D3 early in infection and that early events in viral replication are sensitive to inhibitors of some cdks. On the other hand cdk2 is not activated. Here we report studies on the status of members of E2F family in cycling HEp-2 and HeLa cells and quiescent serum-starved, contact-inhibited human lung fibroblasts. The results show that (i) at 8 h postinfection or thereafter, E2F-1 and E2F-5 were posttranslationally modified and/or translocated from nucleus to the cytoplasm, (ii) E2F-4 was hyperphophorylated, and (iii) overall, E2F binding to cognate DNA sites was decreased at late times after infection. These results concurrent with those cited above indicate that late in infection activation of S-phase genes is blocked both by posttranslational modification and translocation of members of E2F family to inactive compartments and by the absence of active cdk2. The observation that E2F were also posttranslationally modified in quiescent human lung fibroblasts that were not in S phase at the time of infection suggests that specific viral gene products are responsible for modification of the members of E2F family and raises the possibility that in infected cells, activation of the S phase gene is an early event in viral infection and is then shut off at late times. This is consistent with the timing of stabilization of cyclin D3 and the events blocked by inhibitors of cdks.
Insights
Herpes simplex virus 1 infection alters E2F proteins, blocking S-phase gene activation by modifying and relocating E2F and inhibiting cdk2. This suggests viral control over the cell cycle for replication.
Area of Science:
- Cell Biology
- Virology
- Molecular Biology
Background:
- Cell cycle progression from G1 to S phase relies on cyclin-dependent kinases (cdk4 and cdk2) phosphorylating retinoblastoma protein, releasing E2F.
- E2F transcription factors drive S phase and cell cycle gene expression.
- Herpes simplex virus 1 (HSV-1) stabilizes cyclin D3 early in infection, and viral replication is sensitive to cdk inhibitors, yet cdk2 remains inactive.
Purpose of the Study:
- To investigate the status of E2F family members in HSV-1 infected cells.
- To understand how HSV-1 affects E2F function and S-phase gene activation.
Main Methods:
- Studied E2F family members (E2F-1, E2F-4, E2F-5) in cycling HEp-2 and HeLa cells, and quiescent human lung fibroblasts after HSV-1 infection.
- Assessed posttranslational modifications, nuclear-cytoplasmic translocation, and DNA binding of E2F proteins.
Main Results:
- HSV-1 infection led to posttranslational modification and/or nuclear-to-cytoplasmic translocation of E2F-1 and E2F-5 starting at 8 hours post-infection.
- E2F-4 became hyperphosphorylated.
- Overall E2F DNA binding decreased at later infection times.
- E2F modification also occurred in quiescent cells, suggesting viral gene product involvement.
Conclusions:
- Late in HSV-1 infection, S-phase gene activation is inhibited by E2F modification, translocation, and the lack of active cdk2.
- Viral gene products likely mediate E2F modification, potentially controlling S-phase gene activation as an early viral replication event that is later suppressed.