Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species

Abstract

The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G 2 phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G 2 phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1-and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity.

Molecular Pathways: Rodent Parvoviruses—Mechanisms of Oncolysis and Prospects for Clinical Cancer Treatment

“…1). H-1PV has been reported to induce apoptotic cell death in various cancer cell lines (27,(31)(32)(33), and this is reflected in the statistically very significant interaction of PVs with components of the apoptosis-regulating pathway (Fig. 2).…”

“…Arrested cells are proposed to offer a more favorable terrain for PV replication. Of interest, expression of the NS1 protein alone is sufficient to induce cell-cycle arrest (27,29,30), in keeping with the fact that NS1 is the major effector of virus cytotoxicity.…”

“…PV-induced DDR signaling is mediated by activation of ATM but not ATR; is characterized by phosphorylation of H2AX, NBS1, RPA32, CHK2, and P53; and results in accumulation of DNA repair proteins in viral replication centers (16,17). Consequently, cell-cycle arrest after PV infection is observed at the S-G 2 transition or G 2 phase depending on the virus and cell type (27,28). It is associated with upregulation of cell-cycle control proteins such as CDK1, CCNB1/cyclinB1, CDKN1A/p21/Cip1, CDKN1B/p27/Kip1, and RLB2/p130 (27,29,30).…”

“…Consequently, cell-cycle arrest after PV infection is observed at the S-G 2 transition or G 2 phase depending on the virus and cell type (27,28). It is associated with upregulation of cell-cycle control proteins such as CDK1, CCNB1/cyclinB1, CDKN1A/p21/Cip1, CDKN1B/p27/Kip1, and RLB2/p130 (27,29,30). Arrested cells are proposed to offer a more favorable terrain for PV replication.…”

“…Stalled replication forks (25) and ssDNA genomes of PV-related adeno-associated viruses (26) are known to activate the DDR. Another DDRactivating mechanism triggered by PVs is NS1-induced production of reactive oxygen species (ROS) causing DNA damage (27). PV-induced DDR signaling is mediated by activation of ATM but not ATR; is characterized by phosphorylation of H2AX, NBS1, RPA32, CHK2, and P53; and results in accumulation of DNA repair proteins in viral replication centers (16,17).…”