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Three regions of the pRB pocket domain affect its inactivation by human papillomavirus E7 proteins
Frederick A Dick1, Nicholas J Dyson
1Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA.
Abstract:
A critical event in papillomavirus transformation of human cells is the inactivation of pRB by the E7 protein. E7, like many other viral oncoproteins, possesses a well-characterized LXCXE peptide motif that interacts with the pocket domain of pRB. Disruption of the LXCXE-binding cleft on pRB renders it resistant to E7 binding and inactivation. Such binding cleft mutants of pRB are capable of inducing a G(1) arrest in the human papillomavirus 18-transformed HeLa cell line. We show here that the efficient inactivation of pRB in HeLa cells does not simply depend on the integrity of the LXCXE-binding cleft. Multiple site-directed mutants that alter conserved surfaces of the pRB pocket domain cause HeLa cells to accumulate in G(1). We divide these mutants into two classes: those that can be bound by E7 and those that cannot. The E7 interacting mutants include changes in conserved residues that lie in a groove between the A and B halves of the pocket. Surprisingly, none of these mutants show a clear defect in any of the known mechanisms for pRB inactivation by E7. Analysis of mutants that are compromised for E7 binding reveals that this interaction depends on both the LXCXE-binding cleft and on a conserved group of lysines adjacent to the cleft. These basic amino acids on pRB define a discrete interaction point with E7. These residues most likely form ionic interactions with conserved acidic amino acids on E7 since a stable pRB/E7 interaction was restored when the lysine residues on pRB and the acidic residues on E7 were interchanged.
Insights
Human papillomavirus E7 protein inactivates pRB tumor suppressor. This study reveals E7 binding to pRB involves more than the LXCXE motif, identifying key lysine residues for stable interaction and cell cycle arrest.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Human papillomavirus (HPV) oncoproteins, like E7, are crucial for cellular transformation.
- The E7 protein targets the retinoblastoma tumor suppressor protein (pRB) for inactivation.
- E7 interacts with pRB via a specific LXCXE peptide motif within the pRB pocket domain.
Purpose of the Study:
- To investigate the mechanisms of pRB inactivation by the HPV E7 protein.
- To identify additional interaction sites between E7 and pRB beyond the canonical LXCXE motif.
- To understand how mutations in pRB affect E7 binding and subsequent cellular effects.
Main Methods:
- Site-directed mutagenesis of the pRB pocket domain.
- Analysis of pRB mutants for E7 binding affinity.
- Assessment of pRB mutant-induced G(1) cell cycle arrest in HeLa cells.
- Investigating the role of conserved residues and charged amino acids in pRB-E7 interaction.
Main Results:
- Mutations in pRB's LXCXE-binding cleft render it resistant to E7 inactivation, causing G(1) arrest.
- Multiple pRB mutants, altering conserved pocket surfaces, also induce G(1) arrest, independent of simple LXCXE cleft integrity.
- E7 binding to pRB requires both the LXCXE-binding cleft and adjacent conserved lysine residues, forming a distinct interaction site.
- Mutations compromising E7 binding highlight the importance of these lysine residues and their potential ionic interactions with acidic residues on E7.
Conclusions:
- pRB inactivation by E7 is a complex process not solely dependent on the LXCXE-binding cleft.
- A conserved cluster of lysine residues on pRB, adjacent to the LXCXE cleft, is critical for stable E7 interaction.
- This interaction likely involves ionic bonds between pRB lysines and E7 acidic residues, contributing to pRB inactivation and cell cycle control evasion.