The ATR DNA damage response promotes productive replication of HPV through elevation of dNTPs
Text by Cary Moody and Dan Anacker
The importance of the ATM-dependent DNA damage response (DDR) in the facilitating productive replication of HPV has been well documented over the past several years. However, in this study we explored the contribution of the ATR-dependent DDR to HPV replication, a pathway that is normally activated in response to replication stress. We demonstrate that HPV31 activates the ATR/Chk1/E2F1 signaling pathway in an E7-dependent manner to elevate levels of RRM2, a key factor in the de novo synthesis of dNTPs. Importantly, we found that RRM2 is necessary to provide dNTPs for productive viral replication. The utilization of the ATR/Chk1/E2F1 pathway is significant because productive viral replication takes place in differentiating epithelial cells that are arrested in a G2-like environment. Activation of the ATR/Chk1 pathway provides a mechanism by which HPV can gain access to cellular factors outside of S-phase to drive viral replication.
We initiated this project based on the observation that HPV31 positive cells exhibit high levels of RRM2. Investigation into potential mechanisms of RRM2 regulation led us to several papers demonstrating a link between replication-stress induced ATR/Chk1/E2F1 signaling and elevation of RRM2 to provide dNTPs for DNA repair. ATR activity had recently been shown to be required for productive viral replication, so we investigated if ATR/Chk1/E2F1 contributed to viral replication through upregulation of RRM2. Surprisingly, we found both E2F1 and RRM2 protein levels increased dramatically 24-48 hours post-differentiation in an ATR/Chk1-dependent manner. This was particularly exciting because our results suggested that E7-induced cell cycle re-entry upon differentiation results in replication stress and activation of ATR/Chk1. HPV then utilizes this pathway to maintain E2F1 levels and elevate RRM2 upon differentiation, providing dNTPs for viral genome amplification.
The biggest challenge encountered during this research was linking RRM2 upregulation to dNTP production and late viral events. Using organotypic raft cultures derived from HPV31 positive epithelial cells, we identified a subset of differentiating cells exhibiting high levels of RRM2 that were also positive for cellular markers of productive replication. RRM2 knockdown decreased dNTP pools in differentiating cells and blocked viral genome amplification, highlighting the importance of RRM2 in providing dNTPs for viral DNA synthesis. Working on this project was an exciting new endeavor for our lab since it has opened up new areas of research, specifically HPV manipulation of the ATR DDR pathway and nucleotide metabolism. These topics will provide interesting questions for future research.
This model (figure 8 from the article) shows how HPV utilizes the ATR/Chk1 signaling pathway to increase RRM2 levels and provide dNTPs for productive replication.
Introducing the authors
Dr. Cary Moody and Dr. Dan Anacker from the University of North Carolina at Chapel Hill, USA
About the research
HPV31 utilizes the ATR-Chk1 pathway to maintain elevated RRM2 levels and a replication-competent environment in differentiating Keratinocytes
Daniel C. Anacker, Heather L. Aloor, Caitlin N. Shepard, Gina M. Lenzi, Bryan A. Johnson, Baek Kim, Cary A. Moody
Virology, Volume 499, December 2016, Pages 383–396