Novel recombinant papillomavirus genomes expressing selectable genes

Abstract

Papillomaviruses infect and replicate in keratinocytes, but viral proteins are initially expressed at low levels and there is no effective and quantitative method to determine the efficiency of infection on a cell-to-cell basis. Here we describe human papillomavirus (HPV) genomes that express marker proteins (antibiotic resistance genes and Green Fluorescent Protein), and can be used to elucidate early stages in HPV infection of primary keratinocytes. To generate these recombinant genomes, the late region of the oncogenic HPV18 genome was replaced by CpG free marker genes. Insertion of these exogenous genes did not affect early replication, and had only minimal effects on early viral transcription. When introduced into primary keratinocytes, the recombinant marker genomes gave rise to drug-resistant keratinocyte colonies and cell lines, which maintained the extrachromosomal recombinant genome long-term. Furthermore, the HPV18 “marker” genomes could be packaged into viral particles (quasivirions) and used to infect primary human keratinocytes in culture. This resulted in the outgrowth of drug-resistant keratinocyte colonies containing replicating HPV18 genomes. In summary, we describe HPV18 marker genomes that can be used to quantitatively investigate many aspects of the viral life cycle.

Persistence of an Oncogenic Papillomavirus Genome Requires cis Elements from the Viral Transcriptional Enhancer

“…Cells cotransfected with the wild-type HPV18 genome and the pCpGneo URR (7112) plasmid gave rise to robust colonies after selection in G418. We showed previously that the formation of robust, expanding colonies reflects stable maintenance replication of recombinant HPV18 marker genomes ( 29 ). Therefore, primary keratinocytes were cotransfected with the wild-type HPV18 genome and the series of pCpGneo replicons shown in Fig.…”

“…3A ). We previously showed that the Neo R expression cassette derived from this plasmid can be inserted into the late region of the HPV18 genome (replacing the ΔLR deletion described above) to generate a “marker genome” ( 29 ). These marker genomes are able to persistently and stably replicate in keratinocytes, demonstrating that the vector components are not detrimental to viral DNA replication and persistence ( 29 ).…”

“…A replicon lacking a substantial part of the L1 and L2 open reading frames was maintained very efficiently, even without complementation. This led to the development of HPV18-derived marker genomes that encoded selectable markers (neomycin resistance, zeocin resistance, and green fluorescent protein) inserted into the late region ( 29 ). Not surprisingly, replicons with the URR deleted were unable to replicate, even when complemented.…”

Human papilloma virus: An etiological and prognostic factor for oral cancer?

“…HPV is also known to interact with the tetraspanins CD63 and CD151 to gain cellular entry . After infection, the HPV viral genome must amplify to a low copy number as stable or episomal extra chromosomal elements . The episomal form acts as a reservoir in infected cells, which are morphologically indistinguishable from non‐infected cells, and is responsible for the latent status of infection .…”

The Promise of Proteomics in the Study of Oncogenic Viruses

“…differentiation). Quasivirus particles (recircularized viral genomes packaged in a cell line overexpressing the capsid proteins) can be used to generate papillomavirus and polyomavirus particles to study the early stages of infection, and in theory epitope tagged versions of viral proteins could be packaged in these recombinant particles to facilitate their detection and localization (33,34). More efficient methods are also being established to induce the late stages of infection.…”