An Interaction with PARP-1 and Inhibition of Parylation Contribute to Attenuation of DNA Damage Signaling by the Adenovirus E4orf4 Protein

Abstract

The adenovirus (Ad) E4orf4 protein was reported to contribute to inhibition of ATM- and ATR-regulated DNA damage signaling during Ad infection and following treatment with DNA-damaging drugs. Inhibition of these pathways improved Ad replication, and when expressed alone, E4orf4 sensitized transformed cells to drug-induced toxicity. However, the mechanisms utilized were not identified. Here, we show that E4orf4 associates with the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP-1) and that the association requires PARP activity. During Ad infection, PARP is activated, but its activity is not required for recruitment of either E4orf4 or PARP-1 to virus replication centers, suggesting that their association occurs following recruitment. Inhibition of PARP-1 assists E4orf4 in reducing DNA damage signaling during infection, and E4orf4 attenuates virus- and DNA damage-induced parylation. Furthermore, E4orf4 reduces PARP-1 phosphorylation on serine residues, which likely contributes to PARP-1 inhibition as phosphorylation of this enzyme was reported to enhance its activity. PARP-1 inhibition is important to Ad infection since treatment with a PARP inhibitor enhances replication efficiency. When E4orf4 is expressed alone, it associates with poly(ADP-ribose) (PAR) chains and is recruited to DNA damage sites in a PARP-1-dependent manner. This recruitment is required for inhibition of drug-induced ATR signaling by E4orf4 and for E4orf4-induced cancer cell death. Thus, the results presented here demonstrate a novel mechanism by which E4orf4 targets and inhibits DNA damage signaling through an association with PARP-1 for the benefit of the virus and impacting E4orf4-induced cancer cell death. IMPORTANCE Replication intermediates and ends of viral DNA genomes can be recognized by the cellular DNA damage response (DDR) network as DNA damage whose repair may lead to inhibition of virus replication. Therefore, many viruses evolved mechanisms to inhibit the DDR network. We have previously shown that the adenovirus (Ad) E4orf4 protein inhibits DDR signaling, but the mechanisms were not identified. Here, we describe an association of E4orf4 with the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP-1). E4orf4 reduces phosphorylation of this enzyme and inhibits its activity. PARP-1 inhibition assists E4orf4 in reducing Ad-induced DDR signaling and improves the efficiency of virus replication. Furthermore, the ability of E4orf4, when expressed alone, to accumulate at DNA damage sites and to kill cancer cells is attenuated by chemical inhibition of PARP-1. Our results indicate that the E4orf4–PARP-1 interaction has an important role in Ad replication and in promotion of E4orf4-induced cancer-selective cell death.

NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes

“…In the same vein, activation of sirtuins through resveratrol treatment inhibits adenovirus DNA replication [89, 90]. Another NAD + -dependent enzyme to have been studied in lytic infection is Poly (ADP-Ribose) Polymerase 1 (PARP1); the AdV E4orf4 protein has been found to increase production of viral progeny through inhibition of PARP1, which is activated by the infection-induced DNA damage response (DDR) [91]. PARP-induced synthesis and attachment of long poly (ADP-ribose) chains to proteins has been shown to regulate cellular transcription through chromatin remodeling and modification of transcription factors [92, 93].…”

The roles of PARP-1 and XPD and their potential interplay in repairing bupivacaine-induced neuron oxidative DNA damage

“…Mihaela Robu et al [ 37 ] showed that the damaged DNA-binding protein 2(DDB2), a key lesion recognition protein of the global genomic sub-pathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin. Furthermore, Keren et al [ 38 ] described an association of E4orf4 with the DNA damage sensor PARP-1. E4orf4 reduces phosphorylation of the enzyme and inhibits its activity.…”

En Guard! The Interactions between Adenoviruses and the DNA Damage Response

“…In addition to binding DNA-PK, the Ad E4orf4 protein also associates with PARP-1 [ 103 ]. This association requires the activities of at least two E4orf4 partners: protein phosphatase 2A (PP2A) and PARP itself.…”

“…PARP-1 is recruited to the vicinity of Ad RCs, but recruitment does not require either E4orf4, which is also present in RCs, or PARP activity. E4orf4 was shown to inhibit parylation that was stimulated both by chemically induced DNA damage and by Ad infection, and this inhibition required the interaction of E4orf4 with its major partner, PP2A [ 103 ]. Indeed, PARP-1 appeared to be hypophosphorylated in the presence of E4orf4, and this state of reduced phosphorylation likely contributed to PARP-1’s diminished activity, as phosphorylation of various residues was shown to enhance parylation by PARP-1 [ 154 , 155 ].…”

“…Thus, restraining PARP can contribute to the Ad life cycle both by allowing E4orf4 to inhibit ATM and ATR signaling, which contributes to the efficiency of Ad replication [ 102 ], and by inhibiting additional PARP-regulated processes. Indeed, attenuation of PARP enhanced the efficiency of replication of an E4-mutant Ad [ 103 ], which is normally deficient in replication, at least in part because of its inability to inhibit several DDR branches.…”