Novel recombinant papillomavirus genomes expressing selectable genes

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• Papillomaviruses infect keratinocytes, but quantifying early infection efficiency is challenging due to low initial viral protein expression.
• Existing methods lack the sensitivity and quantitative capability for cell-to-cell infection efficiency determination.

Purpose of the Study:

• To develop recombinant human papillomavirus (HPV) genomes expressing marker proteins for quantitative analysis of early HPV infection stages.
• To establish a method for elucidating the early events in HPV infection of primary keratinocytes on a cell-to-cell basis.

Main Methods:

• Recombinant HPV18 genomes were engineered by replacing the late region with CpG-free marker genes (antibiotic resistance, Green Fluorescent Protein).
• These marker genomes were introduced into primary keratinocytes, assessing their effect on replication and transcription.
• Recombinant HPV genomes were packaged into quasivirions for infection of primary human keratinocytes.

Main Results:

• Insertion of marker genes did not significantly impair early replication or transcription of HPV18 genomes.
• Recombinant HPV genomes were stably maintained extrachromosomally in drug-resistant keratinocyte colonies and cell lines.
• Infection with HPV18 marker quasivirions led to the outgrowth of drug-resistant keratinocyte colonies harboring replicating HPV18 genomes.

Conclusions:

• Novel HPV18 marker genomes provide a quantitative tool for investigating the viral life cycle.
• These tools facilitate detailed studies of early HPV infection dynamics in primary keratinocytes.
• The developed system enables a deeper understanding of papillomavirus infection mechanisms.