In this study, we demonstrated that infection of Bocavirus MVC induces the intra-S-phase arrest to delay S-phase progression and to hijack cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is mediated by ATM signaling through phosphorylation of SMC1. The study also provided evidence that the MVC infection-induced DDR is elicited by replicating viral DNA, which is sensed by the MRN complex. Taken together, the study provides, for the first time, a novel DNA replication model for autonomous parvovirus ().
Fig 10 Proposed model for autonomous parvovirus DNA replication in the context of the intra-S-phase arrest. The proposed pathways utilized by autonomous parvovirus during viral DNA replication are described in detail in the Discussion. The question mark indicates (more ...)
In this model, MVC DNA replication triggers the intra-S-phase arrest through the MRN-ATM-SMC1 pathway. The replicating viral DNA mimics damaged DNA that is sensed by the MRN complex. The intra-S-phase arrest blocks cellular DNA synthesis and therefore prolongs S phase in infected cells, presumably through degradation or transcriptional regulation of DNA replication factors. In contrast, the MRN complex may coordinate DNA replication and repair factors through SMC1 activation to facilitate viral DNA synthesis. The feedback loop between viral DNA replication and the intra-S-phase arrest plays an essential role in modulation of the cellular environment by MVC to make it conducive to viral DNA replication.
One of the important findings of this study is that S phase is required but not sufficient for MVC DNA replication. It has been reported that MVM DNA replication is strictly dependent on cellular replication factors expressed in S phase (58
). The basic replication machinery components, such as PCNA, RPA, pol α, pol δ, and cyclin A, all colocalized within the autonomous parvovirus-associated replication (APAR) bodies (59
). In vitro
studies indicated that the cyclin A level directly affects MVM DNA replication efficiency (56
) and that PCNA, RPA, and pol δ are essential for MVM DNA replication (73
); however, like many other DNA viruses, autonomous parvovirus infection blocks cellular DNA synthesis (43
), which was thought to be due to competition for access to the cellular replication machinery by viral DNA replication (75
). Hence, cellular DNA replication is essential for autonomous parvovirus DNA. Here, we show that MVC DNA replicates poorly in both ATM inhibitor-treated and ATM-knockdown cells which have normal S-phase progression. Thus, we provide evidence that cellular DNA replication is not sufficient for MVC DNA replication. We conclude that, in addition to the requirement that infected cells be in S phase, which supplies DNA replication factors, the intra-S-phase arrest is necessary for autonomous parvovirus to compete with cellular DNA synthesis for viral DNA replication. We hypothesize that the intra-S-phase arrest facilitates the recruitment of DNA replication factors through a DNA repair pathway, since intra-S-phase arrest normally coordinates DNA repair following DDR induced by damaged cellular DNA (25
) and restarts of stalled DNA replication forks (28
Inhibition of cellular DNA replication is a common strategy for DNA viruses to modulate the host cellular environment to make it conducive to viral DNA replication. Due to the limited genetic resource, parvoviruses neither encode their own polymerase nor drive infected cells into S phase through their viral components (75
). In comparison to parvoviruses, the inhibition processes of cellular DNA replication by other DNA viruses are often regulated by viral proteins that target the cellular DNA replication machinery. For instance, via viral protein pUL117, human cytomegalovirus (HCMV) blocks host DNA synthesis by delaying the accumulation of the mini-chromosome maintenance (MCM) complex proteins onto chromatin (41
). Human papillomavirus (HPV) inhibits host DNA replication by viral early protein E4-mediated suppression of cellular replication origin licensing (42
); however, polyomaviruses take advantage of DDR signaling to block cellular DNA synthesis. Simian virus 40 (SV40) infection uses the ATR-Δp53-p21 pathway to downregulate cyclin A-CDK2/1 activity, which forces the host cells to remain in S phase (78
), whereas the polyomavirus RA strain has been shown to utilize ATM-SMC1 signaling to override cell cycle regulation and prolong S phase (79
). As a result, viral infection-triggered intra-S-phase arrest slowed down cellular DNA synthesis; however, the intra-S-phase arrest induced by polyomaviruses is largely regulated by the viral large T antigen (16
). In contrast, none of the MVC-encoded proteins are involved in cell cycle regulation (3
) (). Therefore, we have identified, for the first time, a viral DNA replication-dependent intra-S-phase arrest that is ATM mediated.
The ATM-SMC1 pathway is intimately involved in slowing down the cellular DNA replication rate in response to DSBs (25
); however, how phosphorylated SMC1 interferes with cellular DNA replication remains unclear. At least in the intra-S-phase arrest induced during MVC infection, RFC1, which is a key component of the RFC complex that loads PCNA to replicating DNA (82
), is a target for downregulation (). Notably, during the very early phase of infection, RFC1 colocalized within the viral replication centers and later disappeared from the centers when viral DNA was actively replicating. This led us to hypothesize that RFC1 is required for the conversion of viral ssDNA to the double-stranded replicative form (RF DNA) (, Step 1) upon virus infection. Nevertheless, the downregulation of RFC1 during the intra-S-phase arrest provides a candidate for linking SMC1 activation with downregulation of cellular DNA replication. The function of RFC1 in MVC DNA replication and in SMC1-mediated intra-S-phase arrest warrants further investigation.
Studies of virus infection-induced DDR have uncovered novel mechanisms underlying virus-host interaction (15
). Although early studies indicated that infection by most DNA viruses was able to create lesions on cellular DNA involving viral proteins (44
), whether this is common and the major cause of the DDR signaling is not clear. MVC infection did not cause obvious damage to cellular DNA (); hence, the DNA damage signaling induced during MVC infection must come from viral DNA. We and others previously have shown that replication of autonomous parvovirus is required for triggering a DDR (4
). Here, we provide evidence, for the first time, that replicating viral genomes (or intermediates) mimic damaged DNA (likely DSBs), which, in the case of autonomous parvovirus, likely involves the unique hairpin structures, thereby recruiting the MRN complex and DDR proteins. However, due to the difficulty of isolating such intermediate DNA, we are not able to provide direct evidence to show that such DNA structures can directly induce DDR signaling. Nevertheless, in addition to the fact that the DNA damage sensor, the MRN complex, is directly associated with the replicating viral ssDNA, Nbs1 was phosphorylated in the viral replication centers (), strongly suggesting that a DNA repair pathway followed by the intra-S-phase arrest is involved in MVC DNA replication. Interestingly, accumulating evidence has shown that DNA repair factors localize in the replication compartments of many DNA viruses; for instance, the homologous recombinational repair (HRR) factors are recruited into the replication centers of Epstein-Barr virus (EBV), SV40, and HPV (80
). It was suggested that HRR factors are recruited to repair DSBs on the viral genome in the viral replication compartments but not for viral DNA replication. It is understandable that the DSB-initiated repair pathways of homologous recombination and nonhomologous end joining (NHEJ) are involved in the replication of DNA viruses whose genome is dsDNA, since their replication often involves a step of circularization; however, DNA replication of autonomous parvoviruses, whose genome is ssDNA, follows a rolling-hairpin strategy of DNA replication which does not involve circularization of any replication intermediates (54
). The fact that SMC1, a cohesion protein of chromosome DNA, plays a key role in MVC DNA replication may also suggest that it maintains proper alignment of the parvoviral minichromosome (90
) for terminal resolution of RF DNA (54
), in addition to its role in the intra-S-phase arrest. How these DNA repair factors accumulated in the viral replication centers facilitate viral DNA replication, in particular during autonomous parvovirus infection, remains unknown and is a central question in parvovirus DNA replication.
In summary, MVC infection triggers a MRN-ATM-SMC1-mediated intra-S-phase arrest to create an S-phase environment and to recruit the cellular DNA replication machinery, and perhaps the DNA repair machinery, to facilitate MVC DNA replication. Such a strategy may represent a common feature of the DDR induced by other autonomous parvoviruses, which are dependent on S phase for replication in host cells.