A short HBV RNA region induces RNR-R2 expression in non-cycling cells and in primary human hepatocytes

Abstract

25 Hepatitis B virus infects non-dividing cells in which dNTPs are scarce. HBV replication 26 requires dNTPs. To cope with this constraint the virus induces the DNA damage response 27 (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR 28 holoenzyme, the key regulator of dNTP levels. Previously we reported that the HBx open 29 reading frame (ORF) triggers this pathway. Unexpectedly however, we report here that 30 the production of HBx protein is not essential. We found that a small region of 125 bases 31 within the HBx transcript is sufficient to induce RNR-R2 expression in growth arrested 32 HepG2 cells and in primary human hepatocytes (PHH). The observed HBx embedded 33 regulatory element is named ERE. We demonstrate that ERE is functional as a positive 34 strand RNA polymerase-II transcript. Remarkably, ERE is sufficient to induce the Chk1-35 E2F1-RNR-R2 DDR pathway, previously reported to be activated by HBV. Furthermore, 36 we found that ERE activates ATR but not ATM in eliciting this DDR pathway in 37 upregulating RNR-R2. These findings demonstrate the multitasking role of HBV 38 transcripts in mediating virus-host cell interaction, a mechanism that explains how such a 39 small genome effectively serves such a pervasive virus. 40 41 Author summary 42 The hepatitis B virus (HBV) infects the human liver and over 250 million people 43 worldwide are chronically infected with HBV and at risk for cirrhosis and liver cancer.44 HBV has a very small DNA genome with only four genes, much fewer than other 45 viruses. For propagation the virus consumes dNTPs, the building blocks of DNA, in 46 much higher amounts than the infected cells provide. To cope with this constraint, the 3 47 virus manipulates the cells to increase the production of dNTPs. We found that the virus 48 activates the cellular response to DNA damage upon which the cells increase the 49 production of dNTPs, but instead of repairing cellular DNA, the virus uses them for 50 production of its own DNA. Usually viruses manipulate host cells with one or more of 51 their unique proteins, however the small HBV genome cannot afford having such a 52 unique gene and protein. Instead, we found that here the virus relies on RNA to 53 manipulate the host cells. Our findings highlight the unprecedented principle of a 54 multitasking viral RNA that is not only designed to be translated into proteins but also 55 harbors an independent role in activating the cellular DNA damage response. 56 57 Introduction 58 Hepatitis B virus (HBV) is a non-cytopathic enveloped virus containing a small circular 59 partially double-stranded DNA genome. Upon entering the cell the genome is converted 60 into a covalently closed circular DNA (cccDNA), the viral transcription template [1]. The 61 HBV genome harbors enhancers and promoters regulating the transcription of a number 62 of positive strand transcripts. The generated RNA species are nuclear exported by a 63 unique mechanism that is not entirely understood but requires a RNA region shared by all 64 t...