Development of Keratinocyte Cell Lines Containing Extrachromosomal Human Papillomavirus Genomes.

Human papillomaviruses (HPVs) cause persistent infections in stratified cutaneous and mucosal epithelia. In these infections, the viral DNA replicates as low-copy-number, extrachromosomal, double-stranded-DNA circular plasmids in the nucleus of the dividing basal cells. When the infected cells begin the process of differentiation, the viral DNA amplifies to a high copy number and virions are assembled in the superficial cells. To study HPV DNA replication, our laboratory generates primary keratinocyte cell lines that contain replicating extrachromosomal HPV genomes. Here, we describe protocols to culture human keratinocytes, to transfect viral DNA into cells using electroporation, to determine the efficiency of genome establishment in cells with a colony-forming assay, and to measure the copy number and extrachromosomal status of viral genomes using Southern blotting. These methods can be used to study DNA replication of different oncogenic Alphapapillomavirus HPV types. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Electroporation to transfect keratinocytes with recircularized HPV genomes Alternate Protocol: Use of HPV replicon containing selection marker in keratinocyte transfection Support Protocol 1: Rheinwald-Green method of co-culture of irradiated J2 3T3 feeders and human keratinocytes Support Protocol 2: Recircularization of HPV genomes Basic Protocol 2: Quantitative colony formation assay to measure the efficiency of HPV genome establishment Basic Protocol 3: Southern blot analysis of extrachromosomal viral DNA Support Protocol 3: Hirt extraction of low-molecular-weight DNA Support Protocol 4: Qiagen DNeasy Blood & Tissue DNA extraction Support Protocol 5: Generation of a 32 P-labeled HPV DNA probe.

Regulation of Human Papillomavirus 18 Genome Replication, Establishment, and Persistence by Sequences in the Viral Upstream Regulatory Region.

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the human papillomavirus 18 (HPV18) transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here, we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications for the genome partitioning mechanism of papillomaviruses. IMPORTANCE Persistent infection with oncogenic human papillomaviruses (HPVs) is responsible for ∼5% of human cancers. The viral DNA replicates as an extrachromosomal plasmid and is partitioned to daughter cells in dividing keratinocytes. Using a complementation assay that allows us to separate viral transcription and replication, we provide insight into viral sequences that are required for long-term replication and persistence in keratinocytes. Understanding how viral genomes replicate persistently for such long periods of time will guide the development of antiviral therapies.

Persistent Human Papillomavirus Infection.

Persistent infection with oncogenic human papillomavirus (HPV) types is responsible for ~5% of human cancers. The HPV infectious cycle can sustain long-term infection in stratified epithelia because viral DNA is maintained as low copy number extrachromosomal plasmids in the dividing basal cells of a lesion, while progeny viral genomes are amplified to large numbers in differentiated superficial cells. The viral E1 and E2 proteins initiate viral DNA replication and maintain and partition viral genomes, in concert with the cellular replication machinery. Additionally, the E5, E6, and E7 proteins are required to evade host immune responses and to produce a cellular environment that supports viral DNA replication. An unfortunate consequence of the manipulation of cellular proliferation and differentiation is that cells become at high risk for carcinogenesis.

Hitchhiking of Viral Genomes on Cellular Chromosomes.

Persistent viral infections require a host cell reservoir that maintains functional copies of the viral genome. To this end, several DNA viruses maintain their genomes as extrachromosomal DNA minichromosomes in actively dividing cells. These viruses typically encode a viral protein that binds specifically to viral DNA genomes and tethers them to host mitotic chromosomes, thus enabling the viral genomes to hitchhike or piggyback into daughter cells. Viruses that use this tethering mechanism include papillomaviruses and the gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This review describes the advantages and consequences of persistent extrachromosomal viral genome replication.