Epigenetic Regulation of the Human Papillomavirus Life Cycle

Abstract

Persistent infection with certain types of human papillomaviruses (HPVs), termed high risk, presents a public health burden due to their association with multiple human cancers, including cervical cancer and an increasing number of head and neck cancers. Despite the development of prophylactic vaccines, the incidence of HPV-associated cancers remains high. In addition, no vaccine has yet been licensed for therapeutic use against pre-existing HPV infections and HPV-associated diseases. Although persistent HPV infection is the major risk factor for cancer development, additional genetic and epigenetic alterations are required for progression to the malignant phenotype. Unlike genetic mutations, the reversibility of epigenetic modifications makes epigenetic regulators ideal therapeutic targets for cancer therapy. This review article will highlight the recent advances in the understanding of epigenetic modifications associated with HPV infections, with a particular focus on the role of these epigenetic changes during different stages of the HPV life cycle that are closely associated with activation of DNA damage response pathways.

Keywords: DNA damage response; DNA repair; HPV; epigenetics; histone; life cycle.

Figure 1

Genomic organization and the HPV life cycle. (A) The HPV genome consists of 6–8 open reading frames (ORFs) that are designated by the colored blocks. The early promoter (PE) is located upstream of the E6 ORF, and the late promoter (PL) is located in the E7 ORF. The early polyadenylation site (pAE) is located at the 3’ end of the E5 ORF, and the late polyadenylation site (pAL) is located at the end of the L1 ORF. The E8^E2 transcript is expressed from a promoter located in the E1 ORF (PE8). The Long Control Region (LCR) is an untranslated region that contains the keratinocyte enhancer (KE), origin of replication (ori), E1- (E1BS) and E2-binding sites (E2BS), as well as binding sites for various transcription factors. (B) Uninfected epithelium is shown on the left and HPV-infected epithelium is shown on the right. HPV infects the proliferating basal cells of the stratified epithelium exposed through a microwound. Upon entry, viral genomes are established in the nucleus of infected cells as episomes, early viral genes (E1, E2, E6, E7) are expressed, and the virus quickly amplifies to 50–100 copies per cell in an E1- and E2-dependent manner. HPV episomes are maintained at low-copy number in actively dividing basal keratinocytes by replicating along with cellular DNA. As infected cells divide, one cell remains in the basal layer, whereas the other cell migrates upward and initiates epithelial differentiation. Differentiation triggers the productive phase of the viral life cycle, resulting in viral genome amplification to thousands of copies per cell, late gene expression and virion assembly and release. The early promoter remains active, allowing for continued expression of E6 and E7 in differentiating cells. While differentiation normally results in an exit from the cell cycle, the E6 and E7 proteins deregulate cell cycle control to push differentiating cells back into the cell cycle, providing HPV access to cellular substrates required for productive viral replication. E4 and E5 also contribute to productive viral replication. Expression of L1 and L2 in the uppermost layers of the epithelium results in the assembly and release of virions.

Figure 2

DNA repair-induced epigenetic regulation of the HPV viral life cycle. DDR components shown to play a role in the HPV life cycle are highlighted in red. Upon DNA damage, DSBs can be recognized by the MRN complex (MRE11/Rad50/Nbs1), which, together with TIP60 acetyltransferase, promotes the activation of ATM through phosphorylation (depicted as P) and acetylation (depicted as Ac), respectively. Activated ATM acts as a primary signal to induce a signaling cascade through phosphorylation of histone H2AX on Ser139, forming γH2AX at DNA breaks (depicted as γ). γH2AX promotes the recruitment of various DDR effectors in a highly regulated manner at sites of damage via the binding of scaffolding protein MDC1. MDC1 also recruits the E3 ubiquitin ligase ring finger 8 (RNF8) to initiate K63-linked ubiquitin chains (depicted as U) on the histone linker H1, leading to the recruitment of E3 ubiquitin RNF168. RNF168 specifically catalyzes ubiquitination of H2A/H2AX on lysine 13/15 (H2AK13/15ub): a modification essential for accumulation of 53BP1 as well as the RAP80–BRCA1 complex. High-risk HPV E7 proteins directly interact with RNF168. TIP60 has been shown to block 53BP1 recruitment, which may in turn block NHEJ (non-homologous end-joining) and promote HR repair through the recruitment of BRAC1 and Rad51 to viral chromatin. SIRT1 recruits Nbs1 and Rad51 to HPV chromatin. SCM1 is recruited to HPV genomes in association with CTCF—both of which may contribute to productive replication through recruitment of HR factors. SETD2 mediates trimethylation of histone H3K36 (H3K36me3, depicted as m) to recruit effector proteins to regulate multiple cellular processes, including HR (homologous recombination) repair and alternative splicing, which are processes critical to completion of the HPV life cycle. SETD2-mediated H3K36me3 may facilitate HR repair through the recruitment of LEDGF-CtIP and MRG15-PALB2-BRAC1 to viral chromatin. Additionally, H3K36me3 regulates alternative splicing through recruitment of p52-SRSF1 and MRG15-PTB. ATM activity is also required for H3K36me3 maintenance on viral chromatin through an unknown mechanism. Dashed lines represent links that have not been tested experimentally.