MOLECULAR MECHANISMS OF THE DNA DAMAGE RESPONSE INDUCED DURING PARVOVIRUS INFECTION

Abstract

DNA damage response (DDR) is a critical safeguarding system to protect genomic stability and integrality through a cascade of phosphorylation events of three PI-3-kinase-like kinases: ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Although numerous studies have established that the DDR is mainly triggered by exposure to ultraviolet, ionizing irradiation and chemical treatment, which introduce DNA breaks into genomes, accumulating evidence has demonstrated that infections of most DNA viruses and some retroviruses are able to induce a DDR, which plays a critical role in the life cycle of the viruses. Parvoviruses are small, non-enveloped and single-stranded DNA (ssDNA) viruses, and cause highly contagious diseases that are sometimes fatal in humans and animals. Parvoviral genomes are usually 5-6kb, and are flanked by two inverted terminal repeats. Two parvoviruses, minute virus of canines (MVC), a model virus for the study of human bocavirus that causes respiratory tract diseases in children worldwide, and human parvovirus B19 (B19V), a causative agent of several human diseases including bone marrow failure diseases and hydrops fetalis, were used in our study to probe the mechanisms of parvovirus infection-induced DDR. We found infection of both MVC and B19V triggers phosphorylation of the DDR upstream kinases in their host cells, and MVC mainly activates the ATM signaling pathway, while B19V activates the ATR signaling pathway. Moreover, we identified that inhibition of the kinases through inhibitor treatment or small interfering RNA knockdown significantly blocks viral DNA replication. These results indicate that, in contrast to turn on the protection effects of the DDR, parvovirus activates and hijacks the cellular DDR machinery for viral DNA amplification. Following these studies, we next explored the mechanism by which the ATM signaling pathway contributes to MVC DNA replication. We discovered that MVC infection induces an intra-S phase arrest to block cellular DNA replication and to hijack the DNA replication machinery for viral DNA synthesis. The intra-S phase arrest is dependent on ATM signaling. Moreover, we identified SMC1 (structural maintenance of chromosomes 1) as the key regulator of the viral infection-induced intra-S phase arrest. Either knockdown of SMC1 or complementation with a dominant-negative SMC1 mutant blocked both the intra-S phase arrest and viral DNA replication. Finally, we found that the intra-S phase arrest induced during MVC infection is neither caused by damaged host cellular DNA nor by viral proteins, but by replicating viral genomes, which are physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. Taken together, by using MVC and B19V as model organisms, we have probed the basic mechanism of the DDR induced during parvovirus infection. Our findings have greatly facilitated the understanding of the mechanisms underlying parvovirus DNA replication, and have provided a molecular basis for the novel strategy by which DNA viruses subvert the host cellular DDR signaling to make it conducive for viral DNA replication. Our study also sheds light on the identification of efficient anti-viral targets for the treatment of parvovirus-caused diseases.