The Reservoir of Persistent Human Papillomavirus Infection; Strategies for Elimination Using Anti-Viral Therapies

Abstract

Human Papillomaviruses have co-evolved with their human host, with each of the over 200 known HPV types infecting distinct epithelial niches to cause diverse disease pathologies. Despite the success of prophylactic vaccines in preventing high-risk HPV infection, the development of HPV anti-viral therapies has been hampered by the lack of enzymatic viral functions, and by difficulties in translating the results of in vitro experiments into clinically useful treatment regimes. In this review, we discuss recent advances in anti-HPV drug development, and highlight the importance of understanding persistent HPV infections for future anti-viral design. In the infected epithelial basal layer, HPV genomes are maintained at a very low copy number, with only limited viral gene expression; factors which allow them to hide from the host immune system. However, HPV gene expression confers an elevated proliferative potential, a delayed commitment to differentiation, and preferential persistence of the infected cell in the epithelial basal layer, when compared to their uninfected neighbours. To a large extent, this is driven by the viral E6 protein, which functions in the HPV life cycle as a modulator of epithelial homeostasis. By targeting HPV gene products involved in the maintenance of the viral reservoir, there appears to be new opportunities for the control or elimination of chronic HPV infections.

Keywords: HPV; anti-viral therapy; basal epithelial homeostasis; persistent infection.

Figure 1

Human Papillomavirus Infection of Stratified Epithelium. In healthy epithelial tissue, a proportion of basal keratinocytes retain the potential to proliferate in order to maintain the multilayered epithelial structure. Following HPV infection, viral gene expression can alter the normal differentiation program of the cell to facilitate completion of the productive virus life-cycle, and is tightly regulated in the different epithelial layers. Viral gene expression may in due course trigger a successful immune response, leading to disease regression. It is thought that persistent infection, with limited expression of the viral genome, can occur following regression. It has been suggested that changes in the efficiency of immune surveillance may allow for subsequent reactivation of the disease.

Figure 2

Cell Competition model of Apical Extrusion in Response to Basal Cell Overcrowding [65,66]. Upregulation and translocation of the stress-response protein Piezo-1 stimulate the release of the sphingosine-1-phosphate (S1P) molecule to adjacent cells. The binding of S1P to its receptor leads to induction of the ROCK pathway, with actin cytoskeleton contraction, cell proliferation in the surrounding cells, and cell extrusion. The cell that delaminates from the basal layer has transiently higher expression of the Notch receptor on the cell surface, and becomes a signal-receiving cell Notch pathway signal-receiving cell. p53 is one of the transcriptional regulators of the Notch precursor protein. High-risk E6 protein interacts with E6AP and targets p53 for proteasome degradation, downregulating the total Notch receptor expression level. Low-risk E6 proteins can inhibit p300 and alter p53-dependent gene transcription. Cutaneous low-risk E6 protein binds with MAML to inhibit Notch-pathway activation. ROCK can also be activated by a non-canonical Notch signalling pathway.

Figure 3

Basal Layer Epithelial Homeostasis in the Presence or Absence of hrHPV E6 Protein. (A) In the uninfected basal layer, keratinocytes exit the cell cycle (at confluence) in response to contact inhibition signals, with a small proportion of cells retaining their proliferative potential. Keratinocytes that stochastically commit to differentiate and delaminate from the basal layer and are extruded into the upper layers where they express keratinocyte differentiation, such as keratin 10 (K10). (B) The expression of viral E6 protein confers a preferential advantage over neighbouring uninfected cells with regard to basal layer retention. HPV-infected keratinocytes (expressing E6, orange) will gradually displace uninfected cells (white) as a result of their altered response to the local cellular environment. (C) As cell density increases, keratinocytes exit the cell cycle and express the differentiation marker (e.g., K10). The proportion of cells in cycle can be measured using cell cycle markers (geminin, Ki67, or MCM). As cell density increases, E6-expressing cells remain in cycle and differentiation (K10 expression) is inhibited. The dashed circle represents the point at which the cultured keratinocytes reach full confluency. (D) The images show two fluorescently tagged cell lines immediately after plating, and again seven days later. The upper images reveal the equivalent growth of red and green tagged normal keratinocytes (eGFP)/(mCherry). The lower images reveal how the preferential growth advantage of cells expressing E6 is manifest over the seven-day growth period (eGFP)/HPV16E6(mCherry). Cells expressing HPV16E6 are preferentially retained in the lower layer of cells and displace normal keratinocytes.