HPV31 utilizes the ATR-Chk1 pathway to maintain elevated RRM2 levels and a replication-competent environment in differentiating Keratinocytes

Abstract

Productive replication of human papillomaviruses (HPV) is restricted to the uppermost layers of the differentiating epithelia. How HPV ensures an adequate supply of cellular substrates for viral DNA synthesis in a differentiating environment is unclear. Here, we demonstrate that HPV31 positive cells exhibit increased dNTP pools and levels of RRM2, a component of the ribonucleotide reductase (RNR) complex, which is required for de novo synthesis of dNTPs. RRM2 depletion blocks productive replication, suggesting RRM2 provides dNTPs for viral DNA synthesis in differentiating cells. We demonstrate that HPV31 regulates RRM2 levels through expression of E7 and activation of the ATR-Chk1-E2F1 DNA damage response, which is essential to combat replication stress upon entry into S-phase, as well as for productive replication. Our findings suggest a novel way in which viral DNA synthesis is regulated through activation of ATR and Chk1 and highlight an intriguing new virus/host interaction utilized for viral replication.

Modulation of the DNA damage response during the life cycle of human papillomaviruses

“…(55, 56). Numerous studies have demonstrated that HPV exploits both the ATM and ATR DDR pathways in order to successfully replicate its genome (8, 12, 57). In contrast, little is known regarding the importance of HPV’s interaction with the DNA-PK pathway for the viral life cycle.…”

“…Activation of the ATR/Chk1 pathway is also required for the stable maintenance of viral episomes (8, 12, 14), and recent studies have found that ATR/Chk1 activation is also required for productive viral replication (8, 12). Both E7 and E1 can independently activate the ATR/Chk1 pathway (8, 82, 87).…”

“…Activation of the ATR/Chk1 pathway in response to replication stress induces a re-wiring of E2F signaling, resulting in increased expression of genes involved in DNA repair and replication to prevent DNA damage and allow for survival (104). Recent studies from our lab have shown that HPV31 uses the ATR/Chk1 pathway to increase expression of the small subunit of the ribonucleotide reductase complex, RRM2, in an E7- and E2F1-dependent manner (12). RRM2 is required for the de novo synthesis of dNTPs and is necessary for the maintenance of nucleotide pools for productive replication in differentiating HPV31 positive cells (12, 105).…”

“…Numerous studies support a role for the DDR in facilitating HPV replication, with activation of the ATM and ATR pathways being required for productive viral replication (8–12). Activation of the ATR pathway may also contribute to the establishment of HPV genomes (13), as well as to the stable maintenance of viral genomes in undifferentiated cells (8, 12, 14). Three licensed HPV vaccines offer great promise in reducing the number of deaths due to cervical cancer, however these vaccines are not therapeutic.…”

Checkpoint Kinase 1 Is Overexpressed during HPV16-Induced Cervical Carcinogenesis

“…Checkpoint kinase 1 (Chk1) is one of the key regulators in the signal transduction pathway set in motion in response to DDR, which activates different cell cycle checkpoints including G1-S, intra-S, G2-M, and mitotic spindle checkpoints, contributing to the maintenance of the genomic stability. Significant progress has been made toward the understanding of Chk1 regulation and its implications in viral infection, cancer etiology, and therapy [19][20][21]. Chk1 has been found to be overexpressed in a variety of human malignancies, including colon, liver, breast, and gastric carcinoma, etc.…”