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

Abstract

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. INTRODUCTIONEukar yotic cells employ multiple strategies of checkpoint signaling and DNA repair mechanisms to monitor and repair damaged DNA [1][2][3][4][5] . There are two branches of the checkpoint response pathway, ataxia telangiectasia-mutated (ATM) pathway and ATM-Rad3-related (ATR) pathway. The major difference between ATM and ATR is the type of DNA damage to which each responds. For example, ATM responds to ionizing radiation (IR) and other agents that cause double-strand breaks (DSBs) in DNA. ATR responds to ultraviolet radiation (UV) radiation and other agents that induce the accumulation of stalled replication forks and subsequent single-stranded breaks (SSBs) in DNA. The DSBs are recognized by the Mre11-Rad50-Nbs1 complex, which recruits and activates ATM kinase [6] . The SSBs are coated 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

At a Crossroads: Human DNA Tumor Viruses and the Host DNA Damage Response

“…Moreover, HBV infection increased survival of UV-treated (ATR-activated) cells but not γ-irradiated (ATM-activated) hepatocytes [104,105]. These data suggest that HBV replication, while activating ATR, also perturbs downstream signaling to ensure infected cell survival.…”

Viral manipulation of DNA repair and cell cycle checkpoints

“…HIV-Vpr initiates ATR/Chk1 signaling, perhaps due to HIV-Vpr-induced replication stress, which activates the G2/M checkpoint and promotes HIV infection [56,57]. While HBV-HBx protein initiates ATR signaling that is important for efficient HBV replication [58], it then abrogates downstream ATR-mediated checkpoint signaling [59], and cells expressing HBV-HBx continue through G2/M into mitosis [60]. Thus, similar to EBV manipulation of the ATM pathway, HBV activates ATR damage signaling but then disables downstream ATR-mediated checkpoint signaling.…”

“…EBV-infected cells fail to accumulate p21 in response to DNA damaging agents even though p53 expression and damage-induced phosphorylation is unaffected [128]. HBV, while activating ATR and downstream Chk1 phosphorylation, inhibits downstream signaling via degradation of p21, promoting HBV replication [59]. HPV-E7 promotes Cdk2 activity via upregulation of Cdc25A, which is a Cdk2 activator [129].…”

DNA Damage Sensor γ‐H2AX Is Increased in Preneoplastic Lesions of Hepatocellular Carcinoma

“…For example, HBV has been reported to directly regulate the DNA damage response in host cells [6]. HBV stimulates ATM- and Rad3-related protein kinase (ATR)andcheckpoint kinase 1 (Chk1) pathways [7], leading to the acquisition of strengthened survival against DNA damage. Moreover, HBV X gene product (HBX), widely recognized as a possible viral carcinogen [8, 9], plays a critical role in the phosphorylation and inactivation of Rb via activating p38 mitogen-activated protein kinase [10].…”