Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection.

AIM: To explore whether acute cellular DNA damage response is induced upon hepatitis B virus (HBV) infection and the effects of the HBV infection. METHODS: We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chk1 phosphorylation foci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet (UV) radiation and ionizing radiation (IR)-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells. RESULTS: We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chk1, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h after HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increased apoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection. CONCLUSION: HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.

Cellular DNA repair cofactors affecting hepatitis B virus infection and replication.

AIM: To investigate whether hepatitis B virus (HBV) infection activates DNA damage response and DNA repair cofactors inhibit HBV infection and replication. METHODS: Human hepatocyte cell line HL7702 was studied. Immunoblotting was performed to test the expression of ataxia telangiectasia-mutated (ATM)-Rad3-related protein (ATR), p21 and the level of phosphorylation of Chk1, p53, H2AX, ATM in HBV-infected or non-infected-cells. Special short RNAi oligos was transfected to induce transient ATR knockdown in HL7702. ATR-ATM chemical inhibitors caffeine (CF) and theophylline (TP), or Chk1 inhibitor 7-hydroxystaurosporine (UCN01) was studied to determine whether they suppress cellular DNA damage response and MG132 inhibits proteasome. RESULTS: The ATR checkpoint pathway, responding to single-strand breaks in DNA, was activated in response to HBV infection. ATR knockdown cells decreased the HBV DNA yields, implying that HBV infection and replication could activate and exploit the activated DNA damage response. CF/TP or UCN01 reduced the HBV DNA yield by 70% and 80%, respectively. HBV abrogated the ATR-dependent DNA damage signaling pathway by degrading p21, and introduction of the p21 protein before HBV infection reduced the HBV DNA yield. Consistent with this result, p21 accumulation after MG132 treatment also sharply decreased the HBV DNA yield. CONCLUSION: HBV infection can be treated with therapeutic approaches targeting host cell proteins by inhibiting a cellular gene required for HBV replication or by restoring a response abrogated by HBV, thus providing a potential approach to the prevention and treatment of HBV infection.