Functional mapping of the human papillomavirus type 16 E1 cistron

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• Human papillomavirus (HPV) is a common oncogenic virus implicated in cervical and head and neck cancers.
• Replication of the double-stranded, circular HPV genome is dependent on the viral DNA replicase E1 protein.
• Understanding HPV replication mechanisms is crucial for developing antiviral strategies.

Purpose of the Study:

• To characterize the E1 cistron of HPV type 16 (HPV-16), a prevalent oncogenic HPV type.
• To identify the regulatory elements controlling E1 expression and its role in viral DNA replication.
• To elucidate the promoter and enhancer elements essential for HPV-16 genome amplification.

Main Methods:

• Complementation assays were used to assess the necessity of E1 for HPV-16 plasmid synthesis.
• Site-directed mutagenesis was employed to inactivate specific promoter, splice, and enhancer elements.
• Rapid amplification of cDNA ends (RACE) was utilized to map the 5' mRNA ends and define transcription start sites.

Main Results:

• E1 expression was essential for the initial burst of HPV-16 plasmid DNA amplification.
• Mutations in splice donor/acceptor sites and the TATAA box abolished E1 expression.
• A novel promoter, P14, with a transcription start site at HPV-16 nt 14, was identified as responsible for E1 initiation.
• E1 expression was dependent on the HPV-16 keratinocyte-dependent enhancer and modulated by E2 binding sites.

Conclusions:

• P14-initiated E1 expression is critical for and limiting in the initial amplification of the HPV-16 genome.
• The keratinocyte-dependent enhancer and E2 binding sites play significant roles in regulating E1 expression.
• This study provides key insights into the regulation of HPV-16 DNA replication.