Mechanisms by which HPV Induces a Replication Competent Environment in Differentiating Keratinocytes

Abstract

Human papillomaviruses (HPV) are the causative agents of cervical cancer and are also associated with other genital malignancies, as well as an increasing number of head and neck cancers. HPVs have evolved their life cycle to contend with the different cell states found in the stratified epithelium. Initial infection and viral genome maintenance occurs in the proliferating basal cells of the stratified epithelium, where cellular replication machinery is abundant. However, the productive phase of the viral life cycle, including productive replication, late gene expression and virion production, occurs upon epithelial differentiation, in cells that normally exit the cell cycle. This review outlines how HPV interfaces with specific cellular signaling pathways and factors to provide a replication-competent environment in differentiating cells.

Keywords: DNA damage response; HPV; cell cycle; differentiation; replication; virus.

Figure 1

Human papillomavirus (HPV) Life Cycle. HPV infects the basal layer of the stratified epithelium through a microwound. Upon entry into the cell, the virus transiently amplifies to 50–100 copies per cell. HPV genomes are maintained at a stable copy number in undifferentiated basal cells by replicating along with cellular DNA. Upon differentiation, the productive phase of the life cycle is activated, resulting in late gene expression and amplification of viral genomes to thousands of copies per cell. The expression of E6 and E7 allows for cell cycle re-entry upon differentiation, providing cellular factors for productive replication. E4 and E5 also contribute to efficient productive replication. Expression of L1 and L2 promotes the encapsidation of newly replicated genomes, resulting in virion release from the uppermost layers of the epithelium (brown hexagons).

Figure 2

Linear depiction of the HPV31 genome. The open reading frames (ORF) are indicated by the color blocks. The early promoter is located upstream of the E6 ORF (p97) and the late promoter is located in the E7 ORF (p742). E8^E2 is expressed from a promoter located in the E1 ORF (pE8). The early polyadenylation site is located at the 3’ end of the E5 ORF (pAE) and the late polyadenylation site (pAL) is located in the URR (Upstream Regulatory Region). The origin of replication, as well as E1 and E2 binding sites are also located in the URR.

Figure 3

Cellular factors targeted by HPV proteins to maintain cell cycle competency in suprabasal cells. E7 pushes differentiating cells back into the cell cycle by binding to pRb, p107 and p130, which regulate entry into S-phase by negatively regulating E2F transcription factors. Disruption of the pRb/E2F interaction by E7 allows for constitutive activation of E2F-responsive genes, allowing for S-phase re-entry by post-mitotic cells. Unscheduled S-phase entry induced by E7 results in increased p53 that is targeted for degradation by E6 to avoid apoptosis or cell cycle arrest in G1, as well as to block p53’s negative effects on productive replication. E5 contributes to productive viral replication by maintaining cell cycle competency upon differentiation through interaction with BAP31, as well as through activation of epidermal growth factor receptor (EGFR), mitogen activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK)1/2. E4 may increase the efficiency of viral genome amplification by sustaining a G2-arrested environment upon differentiation, and through activation of MAPK signaling (p38, ERK1/2, pJNK). T bars indicate inhibition. Arrows indicate activation. HDAC: histone deacetylase; PCNA: proliferating cell nuclear antigen.

Figure 4

Schematic of the Ataxia-Telangiectasia Mutated (ATM), DNA-dependent Protein Kinase (DNA-PK), and ATM and Rad3-related (ATR) DNA damage response pathways. ATM and DNA-PK are activated in response to double strand DNA breaks (DSBs). ATM facilitates DNA repair through high-fidelity homologous recombination (HR), however, DNA-PK promotes repair through error-prone non-homologous end joining (NHEJ). DNA-PK is activated by the DNA damage sensor complex of Ku70/Ku80, while ATM is activated by the DNA damage sensor complex MRN (Mre11, Rad50, Nbs1) and the TIP60 acetyltransferase. ATM phosphorylates numerous downstream effectors, including H2A.X (gH2AX), Chk2, p38, p53, SMC1 and Breast Cancer Gene 1 (BRCA1) to induce cell cycle arrest and facilitate DNA repair, or to promote apoptosis in the case of severe DNA damage. ATR is activated by single-stranded DNA (ssDNA) generated by replication stress or the resection of DSBs. ssDNA is protected by the tripartite complex RPA, which promotes ATR activation through recruitment of ATRIP, a critical ATR regulator. The Rad17/RFC complex also binds to RPA-coated ssDNA and loads the 9-1-1 complex (Rad9-Hus1-Rad1). 9-1-1 recruits TOPBP1, which is necessary for ATR activation. Claspin mediates the activation of Chk1 by ATR, leading to the replication stress response. T bars indicates inhibition. Arrows indicate activation.

Figure 5

Modulation of the ATM and ATR DNA damage response pathways to promote productive viral replication. HPV-induced activation of ATM requires the STAT5 immune regulator, as well as TIP60, but not the MRN complex. Downstream effectors of ATM required for productive viral replication include the MRN complex, p38/MK2, Chk2, as well as factors involved in homologous recombination repair (Rad51, BRCA1, SMC1). HPV may utilize ATM activity to promote G2 arrest upon differentiation through activities of Chk2, as well as to direct repair to HR on viral genomes through epigenetic modifications and the recruitment of homologous recombination (HR) repair factors. ATR activation in HPV positive cells likely occurs through E7-induced replication stress and requires a STAT5-directed increase in TOPBP1. ATR/Chk1 activation leads to increased levels of E2F1, which drives expression of RRM2, resulting in increased dNTP pools to facilitate productive viral replication.