Experimental Support for Human Papillomavirus Genome Amplification Early after Infectious Delivery

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Human papillomavirus type 16 (HPV16) replication and transcription are extensively studied, but early viral life cycle events remain unclear.
• A lack of efficient infection models has hindered genetic analysis of HPV factors.
• Recent development of an infection model allows for detailed investigation of immediate-early viral events.

Purpose of the Study:

• To investigate HPV16 genome amplification and transcription immediately after viral genome delivery to primary keratinocytes.
• To elucidate the roles of viral proteins E1, E2, and E8^E2 in early HPV16 life cycle events.
• To confirm the utility of a new HPV infection model for studying viral replication and transcription.

Main Methods:

• Utilized a recently developed HPV infection model for primary keratinocyte culture.
• Employed 5-ethynyl-2'-deoxyuridine (EdU) pulse-labeling and fluorescence in situ hybridization (FISH).
• Generated HPV16 mutant viruses with knockouts in E1, E2, and E8^E2 genes to assess their functions.

Main Results:

• HPV16 genome replication and amplification were confirmed to be dependent on viral proteins E1 and E2.
• Knockout of E1 prevented viral genome replication, while knockout of E8^E2 increased genome copy number.
• E2 protein is required for efficient transcription from the early promoter, and E8^E2's main role is controlling genome copy number.

Conclusions:

• The study demonstrates HPV16 genome amplification occurs in an E1- and E2-dependent manner using a novel infection model.
• The viral repressor E8^E2 primarily functions to control viral genome copy number.
• E2 transactivator function is necessary for early promoter activity, and the E8^E2 repressor does not appear to regulate its own promoter.