AbstractHepatitis B virus infects non-dividing cells in which dNTPs are scarce. HBV replication requires dNTPs. To cope with this constraint the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels. Previously we reported that the HBx open reading frame (ORF) triggers this pathway. Unexpectedly however, we report here that the production of HBx protein is not essential. We found that a small region of 125 bases within the HBx transcript is sufficient to induce RNR-R2 expression in growth arrested HepG2 cells and in primary human hepatocytes (PHH). The observed HBx embedded regulatory element is named ERE. We demonstrate that ERE is functional as a positive strand RNA polymerase-II transcript. Remarkably, ERE is sufficient to induce the Chk1-E2F1-RNR-R2 DDR pathway, previously reported to be activated by HBV. Furthermore, we found that ERE activates ATR but not ATM in eliciting this DDR pathway in upregulating RNR-R2. These findings demonstrate the multitasking role of HBV transcripts in mediating virus-host cell interaction, a mechanism that explains how such a small genome effectively serves such a pervasive virus.Author summaryThe hepatitis B virus (HBV) infects the human liver and over 250 million people worldwide are chronically infected with HBV and at risk for cirrhosis and liver cancer. HBV has a very small DNA genome with only four genes, much fewer than other viruses. For propagation the virus consumes dNTPs, the building blocks of DNA, in much higher amounts than the infected cells provide. To cope with this constraint, the virus manipulates the cells to increase the production of dNTPs. We found that the virus activates the cellular response to DNA damage upon which the cells increase the production of dNTPs, but instead of repairing cellular DNA, the virus uses them for production of its own DNA. Usually viruses manipulate host cells with one or more of their unique proteins, however the small HBV genome cannot afford having such a unique gene and protein. Instead, we found that here the virus relies on RNA to manipulate the host cells. Our findings highlight the unprecedented principle of a multitasking viral RNA that is not only designed to be translated into proteins but also harbors an independent role in activating the cellular DNA damage response.