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Direct activation of cyclin-dependent kinase 2 by human papillomavirus E7
Wanxia He1, Doug Staples, Clark Smith
1Genomics-ID, Kalamazoo, Michigan 49006, USA.
Abstract:
Addition of human papillomavirus (HPV) E7 CDK2/cyclin A or CDK2/cyclin E, purified from either insect cells or bacteria, dramatically upregulates histone H1 kinase activity. Activation is substrate specific, with a smaller effect noted for retinoblastoma protein (Rb). The CDK2 stimulatory activity is equivalent in high-risk (HPV type 16 [HPV16] and HPV31) and low-risk (HPV6b) E7. Mutational analyses of HPV16 E7 indicate that the major activity resides in amino acids 9 to 38, spanning CR1 and CR2, and does not require casein kinase II or Rb-binding domain functions. Synthetic peptides spanning HPV16 amino acid residues 9 to 38 also activate CDK2. Peptides containing this sequence that carry biotin on the carboxy terminus, as well as a photoactivated cross-linking group (benzophenone), also activate the complex and covalently associate with the CDK2/cyclin A complex in a specific manner requiring UV. Cross-linking studies that use protein monomers detect association of the E7 peptides with cyclin A but not CDK2. Together, our results indicate a novel mechanism whereby E7 promotes HPV replication by directly altering CDK2 activity and substrate specificity.
Insights
Human papillomavirus (HPV) E7 protein directly enhances CDK2 activity, a key enzyme in cell division. This finding reveals a novel mechanism for how HPV promotes viral replication.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Human papillomavirus (HPV) is a common viral infection.
- The E7 protein of HPV is known to interact with host cell proteins.
- CDK2/cyclin complexes regulate the cell cycle and are implicated in viral replication.
Purpose of the Study:
- To investigate the effect of HPV E7 protein on CDK2 kinase activity.
- To determine the specific regions and mechanisms of E7 interaction with CDK2/cyclin complexes.
- To elucidate the role of E7-mediated CDK2 activation in HPV replication.
Main Methods:
- Purification of HPV E7 protein from insect cells and bacteria.
- Assays to measure histone H1 kinase activity of CDK2/cyclin A and CDK2/cyclin E.
- Mutational analysis of HPV16 E7 protein.
- Synthesis and testing of E7 peptides.
- UV-crosslinking studies to detect E7-protein interactions.
Main Results:
- Addition of HPV E7 protein significantly upregulated histone H1 kinase activity of CDK2/cyclin A and CDK2/cyclin E.
- Activation was substrate-specific, with a lesser effect on retinoblastoma protein (Rb).
- The N-terminal region (amino acids 9-38) of HPV16 E7 was critical for this activity.
- Synthetic peptides corresponding to this region also activated CDK2.
- Cross-linking studies indicated E7 peptides associate with cyclin A but not CDK2.
Conclusions:
- HPV E7 protein directly modulates CDK2 activity and substrate specificity.
- This direct interaction represents a novel mechanism by which E7 promotes HPV replication.
- The findings provide new insights into the molecular pathogenesis of HPV infections.