CCCTC-Binding Factor Recruitment to the Early Region of the Human Papillomavirus 18 Genome Regulates Viral Oncogene Expression

Abstract

Host cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7 and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression.IMPORTANCE The establishment and maintenance of HPV infection in undifferentiated basal cells of the squamous epithelia requires the activation of a subset of viral genes, termed early genes. The differentiation of infected cells initiates the expression of the late viral transcripts, allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity.

Epigenetic regulation of human papillomavirus transcription in the productive virus life cycle

“…Using HPV31 episome-containing cells as a model system, the Laimins group showed that CTCF was predominantly recruited to the late gene region and that depletion of CTCF, or mutation of the L2 binding site cluster resulted in reduced episome copy number and failure of episomal establishment [97]. Somewhat in contrast to these findings, our laboratory has shown that HPV18 genomes have enriched CTCF binding at the high-risk HPV-specific E2 ORF with an absence of binding in the late gene region, suggesting that different high-risk HPV types have evolved different strategies of gene expression control [52,98]. Mutation of the single E2-CTCF binding site in HPV18 had no effect on replication or maintenance of HPV18 episomes, but resulted in increased early transcript production and a concomitant increase in E6 and E7 protein expression and cellular hyperproliferation [98].…”

“…Somewhat in contrast to these findings, our laboratory has shown that HPV18 genomes have enriched CTCF binding at the high-risk HPV-specific E2 ORF with an absence of binding in the late gene region, suggesting that different high-risk HPV types have evolved different strategies of gene expression control [52,98]. Mutation of the single E2-CTCF binding site in HPV18 had no effect on replication or maintenance of HPV18 episomes, but resulted in increased early transcript production and a concomitant increase in E6 and E7 protein expression and cellular hyperproliferation [98]. Importantly, we showed that CTCF-mediated repression of HPV early gene transcription via the stabilization of a chromatin loop formed between the E2 ORF and the URR.…”

“…Similarly, multiple CTCF binding sites have been identified within the genomes of several HPV types. These include a conserved cluster within the late gene region found in over 80% of 125 types screened (high-and low-risk) and also sites within the E2 ORF, present in less than 20% of HPV types analysed and which appears to be conserved in high-risk HPV types only [97,98]. Using HPV31 episome-containing cells as a model system, the Laimins group showed that CTCF was predominantly recruited to the late gene region and that depletion of CTCF, or mutation of the L2 binding site cluster resulted in reduced episome copy number and failure of episomal establishment [97].…”

SAMHD1 regulates human papillomavirus 16 induced cell proliferation and viral replication during differentiation of oral keratinocytes

“…Many excellent studies have been carried out with primary human keratinocytes to characterize the immortalization properties of high risk HPV (HR-HPV) These HPV immortalized cells have proved invaluable at identifying cellular proteins that are required for the life cycle of HR-HPV (3–6, 29–43). For example, it is clear that host cell homologous recombination factors are required for the amplification phase of the viral life cycle (5, 39, 40).…”

Epigenetics and Genetics of Viral Latency

“…For EBV and KSHV, CTCF mediates the recruitment of cohesins and the formation of higher-order DNA conformational structures that have been implicated in regulating the latent to lytic switch, and latency-associated transcription programming. Recent studies with HPV reveal that CTCF regulates the alternative splicing of viral RNA transcripts (Paris et al, 2015), as well as the recruitment of the cohesin subunit SMC1 required for viral genome amplification. CTCF can also bind to the proviral genome of the retrovirus HTLV-1 to regulate RNA splicing, epigenetic boundaries, and the antisense transcription of the viral HBZ gene, necessary for clonal persistence (Satou et al, 2016).…”