Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational constraints affecting E1...

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Human papillomavirus type 16 (HPV16) E1 protein is crucial for viral DNA replication.
• E1 interacts with host factors, including hUBC9 and 16E1-BP, identified via two-hybrid assays.
• hUBC9 is a ubiquitin-conjugating enzyme involved in cell cycle progression; 16E1-BP has an ATPase motif.

Purpose of the Study:

• To generate and characterize HPV16 E1 missense mutants defective in interactions with hUBC9 or 16E1-BP.
• To investigate the functional consequences of these interaction defects on E1's replication activity, ATPase function, and protein-protein interactions.
• To explore the structural and functional modularity of the HPV16 E1 protein.

Main Methods:

• Random mutagenesis of the HPV16 E1 gene to create missense mutants.
• Two-hybrid assays to identify mutants with altered interactions with hUBC9 and 16E1-BP.
• Functional characterization including origin-dependent transient DNA replication assays and ATPase activity measurements.

Main Results:

• Eight HPV16 E1 missense mutants defective for hUBC9 or 16E1-BP interaction were generated.
• Six mutants showed significantly impaired replication; two classes were identified.
• One class (e.g., S330R) was replication-defective but retained most interactions, exhibiting dominant-negative effects. Another class showed multiple interaction defects.
• Single mutations often had pleiotropic effects on E1 functions.

Conclusions:

• HPV16 E1 function is sensitive to mutations affecting hUBC9 and 16E1-BP interactions.
• Replication-defective mutants exhibit distinct interaction and dominant-negative phenotypes.
• The findings suggest that HPV16 E1 domains are not structurally or functionally independent, implying interdependence.