Abstract
Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.
Highlights
These findings led us to investigate whether activation of ATM and/or ATR kinases may result in phosphorylation of the Hepatitis B virus (HBV) core protein (HBc) protein
While there is no TQ motif present in the HBc protein sequence, the C-terminal domain contains three SQ sites involving serine residues at positions 170, 178 and 183 (unless stated otherwise, the positions of amino acids are derived from a 185-aa variant of HBc throughout the study (Figure 1A)
This region of HBc is rich in arginine residues, and was previously shown to be frequently modified by phosphorylation
Summary
Chronic hepatitis (CHB) caused by the Hepatitis B virus (HBV) is a serious liver disease. The World Health Organization (WHO) estimates that in 2019, 296 million people worldwide were chronically infected with HBV. The Hepatitis B virus is a small, enveloped virus with a 3.2 kb long, partially doublestranded circular DNA genome. It encodes only four overlapping open reading frames that are transcribed into 6 RNAs and translated to seven proteins, e.g., pre-core (HBeAg), core (HBc, p21), viral polymerase (P), envelope/surface proteins (S, M and L) and the X protein (HBx) [3,4,5]
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