Interferon treatment of human keratinocytes harboring extrachromosomal, persistent HPV-16 plasmid genomes induces de novo viral integration.

Interferons (IFNs) have been used to treat epithelial lesions caused by human papillomavirus (HPV) persistence. Here, we exposed primary human keratinocytes (HFKs) immortalized by persistently replicating HPV-16 plasmid genomes to increasing levels of IFN-γ. While untreated HFKs retained replicating HPV-16 plasmids for up to 60-120 population doublings, IFN led to rapid HPV-16 plasmid loss.

Analyzing the Human Papillomavirus (HPV) Life Cycle in Primary Keratinocytes with a Quantitative Colony-Forming Assay.

Papillomavirus genomes replicate as extrachromosomal plasmids within infected keratinocytes, requiring the regulated expression of early viral gene products to initially amplify the viral genomes and subvert cell growth checkpoints as part of a complex path to immortalization. Building on contemporary keratinocyte transfection and culture systems, the methods described in this unit form a detailed approach to analyzing critical events in the human papillomavirus (HPV) life cycle, utilizing physiologic levels of viral gene products expressed from their native promoter(s) in the natural host cells for HPV infection. A quantitative colony-forming assay permits comparison of the capacities of various transfected HPV types and mutant HPV genomes to initially form colonies and immortalize human keratinocytes.

DNA nanotherapy for pre-neoplastic cervical lesions.

Objective: This study aims to test the hypothesis that targeted nanoparticle delivery of DNA encoding HPV16-regulated diphtheria toxin (DT-A) will result in the death of HPV16-infected cells.

Materials And Methods: Plasmid constructs containing a HPV16 Long Control Region (LCR) DNA sequence upstream of DT-A or luciferase reporter (Luc) DNA sequences were used to formulate poly(β-amino ester) nanoparticles. The effect on tumor growth of HPV/DT-A-nanoparticle injection directly into HPV16(+) CaSki human cervical cancer cell-derived xenografts in mice was determined.

The truncated C-terminal E2 (E2-TR) protein of bovine papillomavirus (BPV) type-1 is a transactivator that modulates transcription in vivo and in vitro in a manner distinct from the E2-TA and E8^E2 gene products.

The E2 open reading frame of bovine papillomavirus (BPV)-1 encodes a 410 amino acid (aa) transcriptional activator, E2-TA, and collinear polypeptides--E2-TR (243 aa) and E8^E2 (196 aa). E8^E2 and E2-TR share the DNA-binding domain of E2-TA, and both have been defined as transcriptional repressors. Although purified E2-TR and E8^E2 proteins specifically bound E2 sites with similar affinities, only the E2-TR stimulated transcription.

Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers.

Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal.